Method for analyzing collagenous tissues for the detection and diagnosis of bone disease

ABSTRACT

Methods and systems for diagnosing a bone disease related to collagen pathology in a subject are provided. These include providing a bone sample from the subject and determining a quantitative collagen morphology value of the bone sample. A reference value is provided from a non-affected control subject where the reference value is a quantitative collagen morphology value from the same type of bone sample obtained from a population of non-affected control subjects. The quantitative collagen morphology value of the subject&#39;s bone sample is compared to the reference value. If the collagen morphology value is altered versus the reference value, the subject is diagnosed as having a collagen related bone disease. The collagen morphology value can include mean fibril spacings and distributions of the fibril spacings taken from a subject&#39;s bone sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/273,192, filed on Jul. 31, 2009. The entire disclosure of the aboveapplication is incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made under Grant number DE018840 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD

The present disclosure relates to detecting and diagnosing bone diseasesrelating to collagen structure, including osteoporosis and osteogenesisimperfecta in a subject using type I collagen morphology.

INTRODUCTION

This section provides background information related to the presentdisclosure, which is not necessarily prior art.

Bone is an exquisitely evolved biological material with a complexhierarchical structure. At its most fundamental level, bone is atwo-phase composite composed of a relatively soft and ductile collagenmatrix that is impregnated with and surrounded by a much stiffer,stronger reinforcing carbonated apatite phase. Despite the importance ofbone health to overall health, there is a lack of knowledge regardingthe ultrastructure of bone and how mineral and collagen, and theirinteraction with one another, relate to clinically-relevant tissue-leveland structural-level properties. Disease, nutrition, trauma andmechanical loads can impact bone at multiple levels of the tissuehierarchy, including the tissue ultrastructure.

Although many diseases affect bone through changes in mineral and theorganic matrix, osteoporosis is the most common bone disease and is amajor medical and economic burden facing society. Each year, anestimated 1.5 million Americans suffer an osteoporotic fractureresulting in direct-care expenditures of 18 billion dollars a year andoften leading to a downward spiral in quality of life or even death. Anestimated 10 million Americans over the age of 50 have osteoporosis, andanother 34 million are considered at risk. On a more global scale, 75million people in the US, Europe and Japan combined have osteoporosis,and as many as 9 million osteoporotic fractures were reported worldwidein the year 2000. A combination of the aging population coupled with alack of knowledge concerning the material mechanism of the disease andless than ideal diagnostic tools means that the number of osteoporotichip fractures in the United States could triple by the year 2040.

Clinically, osteoporosis is defined in a person as having a bone mineraldensity (BMD) that is 2.5 standard deviations below the peak bone massof the average healthy person of the same gender, as measured bydual-energy x-ray absorptiometry (DEXA). Although popular and widelyused, DEXA has several limitations. Most significantly, standard DEXAuses a two-dimensional (2D) projection to measure areal BMD (g/cm²)versus true volumetric BMD (g/cm³). This 2D measurement is, therefore,dependent on the size and shape of the measured bone and canunderestimate true BMD. Regardless of the method used to measure BMD,estimating fracture risk based solely on BMD is not sufficient. NormalBMD does not guarantee protection from osteoporotic fracture, and manycases of osteoporosis do not become clinically evident until fractureoccurs. Current diagnostic methods for osteoporosis focus exclusively onbone quantity and mineral content and fail to take into account theimportant biological role of collagen matrices.

Another significant bone disease is osteogenesis imperfecta which isbelieved to be caused by genetic defects that affect the body's abilityto make strong bones. In dominant (classical) osteogenesis imperfecta, aperson has too little type I collagen or a poor quality of type Icollagen due to a mutation in one of the type I collagen genes. Inrecessive osteogenesis imperfecta, mutations in other genes interferewith collagen production. The result in all cases is fragile bones thatbreak easily.

It is often, though not always, possible to diagnose osteogenesisimperfecta based solely on clinical features. Clinical geneticists canalso perform biochemical (collagen) or molecular (DNA) tests that canhelp confirm a diagnosis of osteogenesis imperfecta in some situations.These tests generally require several weeks before results are known.Both the collagen biopsy test and DNA test are thought to detect almost90% of all type I collagen mutations. A positive type I collagen studyconfirms the diagnosis of dominant osteogenesis imperfecta, but anegative result could mean that either a collagen type I mutation ispresent but was not detected or the patient has a form of the disorderthat is not associated with type 1 collagen mutations or the patient hasa recessive form of osteogenesis imperfecta. Therefore, a negative typeI collagen study does not rule out osteogenesis imperfecta. When a typeI collagen mutation is not found, other DNA tests to check for recessiveforms are needed and thus prolong confirmatory diagnosis and increasethe time taken for proper diagnosis and screening.

It is, therefore, important to develop quantitative methods to assessthe ultrastructure in bone and other tissues. Such methods couldfacilitate the diagnosis of bone disease and could identify theunderlying mechanism of the disease in order to better focus treatmentand intervention.

SUMMARY

This section provides a general summary of the disclosure and is not acomprehensive disclosure of its full scope or all of its features.

Methods are provided for identifying a subject as a disease candidatewhere the disease affects tissue comprising collagen. A sample of tissuecomprising collagen, such as dentin, bone, tendon, or skin, is providedfrom the subject. A collagen fibril morphology value is determined forthe tissue sample. A reference value, being a reference collagen fibrilmorphology value determined using a tissue sample from a controlsubject, is provided where the tissue sample from the control subject isthe same type of tissue as the tissue sample from the subject. Forexample, determining a collagen fibril morphology value of the tissuesample can include imaging the tissue using atomic force microscopy andmeasuring D-periodic gap/overlap spacing of a collagen fibril or aplurality of collagen fibrils. Also, the reference value can include aD-periodic gap/overlap spacing of a collagen fibril or a plurality ofcollagen fibrils from the control subject. The collagen fibrilmorphology value of the tissue sample is then compared to the referencevalue. The subject is identified as a disease candidate when thecollagen fibril morphology value is different from the reference value.Such methods can use bone tissue to identify a subject as anosteoporosis candidate or an osteogenesis imperfecta candidate.

Further areas of applicability will become apparent from the descriptionprovided herein. The description and specific examples in this summaryare intended for purposes of illustration only and are not intended tolimit the scope of the present disclosure.

DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The drawings described herein are for illustrativepurposes only of selected embodiments and not all possibleimplementations, and are not intended to limit the scope of the presentdisclosure.

FIG. 1. Schematic of two-dimensional fast Fourier transform measurementsin mineralized bone. Panel a shows a representative 3.5 μm×3.5 μmamplitude (error) image that was used to measure the collagen fibrilaxial D-periodicity. The yellow box represents a fibril that was chosenfor measurement. Panel b shows the corresponding 2D FFT from thisfibril. As indicated, the 2D spectrum contains information about theharmonic characteristics of the fibril. The circled peaks are the firstharmonic in the spectrum. The maximum value in this peak corresponds tothe axial repeat distance of the fibril (in this case, 68 nm).

FIG. 2. Three-dimensional rendering of an intracortical region in themurine femur. To produce this figure, a phase image was used as atexture overlay on the corresponding topography (height) image toproduce a rendering of the surface. In this representative 3.5 μm×3.5 μmimage (150 nm height), the rich sample topography characteristic of boneis evident. A bundle of collagen fibrils is seen spanning the surfaceimmediately adjacent to disorganized, woven collagen.

FIG. 3. Axial D-periodic gap/overlap spacings from murine femora. Panela shows the boxplot representation of the axial gap/overlap spacing fromeach of the 5 femurs measured, as well as from all fibrils combined. Thedashed horizontal line indicates the expected 67 nm repeat distance. Foreach sample, the box is the interquartile region (middle 50% of thedata), the horizontal line inside of the box is the median, and thediamond is the mean. The whiskers on the box are the minimum and maximumobservation in each group. When the axial gap/overlap spacings from all193 fibrils are viewed as a histogram with a 1 nm bin size in panel b,there is a population of fibrils near the theoretical 67 nm value.However, the spacing of the fibrils has a distribution ranging from near63 nm to 74 nm. This type of distribution is often overlooked, but mayhave important implications to the development of the anisotropicmechanical behavior of the bone tissue.

FIG. 4. Axial D-periodic gap/overlap spacings from murine femora andovine radii. This figure shows the boxplot representation of the axialgap/overlap spacing from mouse, sham operated sheep and ovariectomized(OVX) sheep. The dashed horizontal line indicates the theoretical 67 nmrepeat distance. When the sham samples were compared to OVX by one-wayANOVA, there was a significant difference present (p=0.048).

FIG. 5. Axial D-periodic gap/overlap spacings from ovine radii. Panel ashows the histogram representation of the axial gap/overlap spacing fromsham operated sheep and ovariectomized (OVX) sheep. When the axialgap/overlap spacings from all fibrils are viewed as histograms with a 1nm bin size, there is a population of fibril spacings in the OVX sheepat 64 nm or below that is almost completely absent from the Sham sheep.Panel b displays the Cumulative Density Function (CDF) calculated fromeach population. The CDF shows what fraction of a given sample iscontained up to a particular value. A Kolmogorov-Smirnov (K-S) testperformed on the two groups indicates that there is a significantdifference in the population distributions of the two groups (p<0.001).

FIG. 6. Schematic representation of the processed mouse femur. Panel ashows a 3D image of a mouse femur, with the anterior surface of thefemur facing the reader (proximal end left, distal end right). The femurwas polished to create a flat intracortical region to image (panel b).Along the length of the femur, 9 locations were analyzed to investigatethe collagen fibril ultrastructure (panel c).

FIG. 7. Representative line scans from each tissue. This figure showsrepresentative 3.5 μm×3.5 μm amplitude images from dentin (A) and bone(B), and a deflection image from tendon (C). The yellow line in eachpanel is where a representative line scan was performed (as shown inpanel D). The line scans in panel D are shown on a normalized heightscale for comparison purposes.

FIG. 8. Schematic representation of two dimensional fast Fouriertransform measurements. Panel a shows a representative 3.5 μm×3.5 μmamplitude image from a bone sample that was used to measure the collagenfibril D-periodicity. The box represents a fibril that was chosen formeasurement. Panel b shows the corresponding 2D FFT from this fibril. Asindicated, the 2D power spectrum contains information about the harmoniccharacteristics of the fibril. The circled peaks are the first harmonicof the spectrum. The maximum value in this peak corresponds to theD-Periodic repeat distance of the fibril.

FIG. 9. D-periodic spacing as a function of axial location in bones.This figure shows the boxplot representation of the D-periodic spacingas a function of axial location in the bone samples. Data from the fourbones were pooled at each location. This figure confirms that there wereno systematic changes in collagen morphology as a function of locationin the bone.

FIG. 10. D-periodic gap/overlap spacings from murine tissues. Panel Ashows the boxplot representation from each sample in each tissue. Therightmost boxplot for each tissue in panel A is the boxplot for allmeasured fibrils within the tissue type. This boxplot is repeated inpanel B. When the groups were compared by one way ANOVA with post hocBonferroni tests, there were no significant differences between any ofthe groups (p-values indicated for each comparison).

FIG. 11. Histogram and cumulative density function of D-periodicspacings from murine tissues. Panel a shows the histogram representationof the D-periodic gap/overlap spacing from dentin, bone, and tendonsamples (1 nm bin size). Panel b displays the cumulative densityfunction (CDF) calculated from each group. The CDF shows what fractionof a given sample is contained up to a particular value. AKolmogorov-Smirnov test performed on the data indicates that there aresignificant differences in the population distributions between all thethree groups.

FIG. 12. Type I Collagen Fibrillar Organization. Collagen is firstsynthesized as a polypeptide chain of amino acids with uninterruptedregions including a repeating Gly-X-Y triplet (X and Y are often prolineand hydroxyproline, respectively). This chain takes a left-handedhelical conformation and then 3 chains come together to form aright-handed triple helix. Once secreted from the cell, non-helicalpro-peptide ends are enzymatically cleaved leaving a 300 nm long, 1.5 nmwide tropocollagen molecule. The molecules self-assemble in a staggered,parallel manner to form a 3D fibril. Because of the space between theends of molecules and the offset from row to row, regions of gaps andoverlaps exist within the fibril and produce an oscillating surfacetopography with a characteristic repeat pattern called the AxialD-periodicity.

FIG. 13. Schematic Representation of Two Dimensional Fast FourierTransform Measurements. Panel a shows a representative 3.5 μm×3.5 μmamplitude image that was used to measure the collagen fibrilD-periodicity. The box represents a fibril that was chosen formeasurement. Panel b shows the corresponding 2D FFT from this fibril. Asindicated, the 2D power spectrum contains information about the harmoniccharacteristics of the fibril. The circled peaks are the first harmonicof the spectrum. The maximum value in this peak corresponds to theD-Periodic repeat distance of the fibril.

FIG. 14. D-Periodic Gap/Overlap Spacings from Murine Femora as aFunction of Axial Location. This figure shows the boxplot representationof the axial gap/overlap spacing from WT (A) and Brt1/+ (B) bones as afunction of axial location. The dashed horizontal line indicates theexpected 67 nm repeat distance. For each sample, the box is theinterquartile region (middle 50% of the data), the horizontal lineinside of the box is the median, and the diamond is the mean. Thewhiskers on the box are the minimum and maximum observation for thatlocation. Qualitatively, there is greater variability between axiallocations in the Brt1/+ bones (B) in comparison to the WT (A) bones.

FIG. 15. D-Periodic Gap/Overlap Spacings from Murine Femora. Boxplotrepresentation of the D-periodic gap/overlap spacing from the 4 bones ineach genotype. The dashed horizontal line indicates the theoretical 67nm repeat distance. When the mean values from the 4 samples in eachgroup were compared by One Way ANOVA, WT and Brt1/+ mice (p=0.392) didnot differ significantly.

FIG. 16. Histogram and Cumulative Density Function of D-PeriodicSpacings from Murine Bone. Panel A shows the histogram representation ofthe D-Periodic gap/overlap spacing from WT and Brt1/+ bones (1 nm binsize). Panel B displays the Cumulative Density Function (CDF) calculatedfrom each group. The CDF shows the fraction of a given sample that iscontained up to a particular value. A Kolmogorov-Smirnov test performedon the data distributions indicates that there is a significantdifferences in the population distributions between WT and Brt1/+ mice(p=0.001).

FIG. 17. Processing and Imaging of the Murine Femur. Panel a shows a 3Dimage of a mouse femur, with the anterior surface of the femur facingthe reader. Nine locations along the length of each femur were analyzedto investigate the collagen fibril ultrastructure (panel b). Panel cshows a large scale image of the bone's surface, indicating a region ofinterest for closer investigation. At this location, a 3.5 μm×3.5 μmimage was obtained and analyzed.

FIG. 18. D-Periodic Gap/Overlap Spacings from Male and Female MurineFemora as a Function of Axial Location. This figure shows the boxplotrepresentation of the D-periodic gap/overlap spacing from Male (A) andFemale (B) bones as a function of axial location. The dashed horizontalline indicates the expected 67 nm repeat distance. For each sample, thebox is the interquartile region (middle 50% of the data), the horizontalline inside of the box is the median, and the diamond is the mean. Thewhiskers on the box are the minimum and maximum observation for thatlocation. Although some statistical differences did exist betweenlocations in each gender (Male: 4 vs. 6; Female: 2 vs. 9, 2 vs. 5, 8 vs.9), there were no systematic differences in either gender.

FIG. 19. Overall D-Periodic Gap/Overlap Spacings from Male and FemaleMurine Femora. This figure shows the boxplot representation of theD-periodic gap/overlap spacing from the 4 bones in each gender. Thedashed horizontal line indicates the theoretical 67 nm repeat distance.When the mean values from the 4 samples in each group were compared byOne Way ANOVA, male and female mice (p=0.564) did not differsignificantly.

FIG. 20. Histogram and Cumulative Distribution Function of D-PeriodicSpacings from Male and Female Murine Bone. Panel A shows the histogramrepresentation of the D-Periodic gap/overlap spacing from Male andFemale bones (1 nm bin size). Qualitatively, there were no differencespresent in the male and female population distributions. Panel Bdisplays the Cumulative Distribution Function (CDF) calculated from eachgroup. The CDF shows the fraction of a given sample that is contained upto a particular value. A Kolmogorov-Smirnov test performed on the datadistributions indicates that there is a no detectable difference in thepopulation distributions between Male and Female mice (p=0.276).

DETAILED DESCRIPTION

The following description of technology is merely exemplary in nature ofthe subject matter, manufacture and use of one or more inventions, andis not intended to limit the scope, application, or uses of any specificinvention claimed in this application or in such other applications asmay be filed claiming priority to this application, or patents issuingtherefrom. A non-limiting discussion of terms and phrases intended toaid understanding of the present technology is provided at the end ofthis Detailed Description.

The present disclosure provides methods, systems, apparatus, andcompositions relating to determining whether a subject has a bonedisease, whether the subject is at risk for developing a bone disease,and for screening a subject to determine whether they have a bonedisease, such as osteoporosis or osteogenesis imperfecta.

Bone evolved by nature to elegantly balance structural and metabolicneeds in the body. Bone health is an integral part of overall health andan understanding of the ultrastructure of healthy bone can help identifyhow disease impacts the nanoscale properties of this biologicalmaterial. The present technology demonstrates that quantitativeassessments of a distribution of Type I collagen fibril morphologies canbe made using atomic force microscopy (AFM). As identified herein,normal bone contains a distribution of collagen fibril morphologies andchanges in this distribution can be directly related to disease state.Specifically, by monitoring changes in the collagen fibril distributionof sham-operated and estrogen-depleted sheep, the present technologydemonstrates the ability to detect estrogen-deficiency-induced changesin Type I collagen in bone. This technology provides new insight intothe ultrastructure of bone as a tissue and the role of materialstructure in bone disease. These observations afford a much-needed invitro procedure that can complement current methods used to diagnoseosteoporosis and other bone diseases.

Type I collagen is the most abundant protein in the body and forms thestructural scaffolding upon which bone is built. The need for accuratequantitative analytical methods to assess collagen's nanoscalemorphology and mechanical integrity with as little disruption to thetissue as possible prompted the present investigation of the collagenultrastructure of bone using atomic force microscopy (AFM). Othermethods successfully used to image the collagen ultrastructure of bone(e.g. transmission and scanning electron microscopy) require the removalof the sample from its natural surroundings followed by harshpreparation techniques including full dehydration and ultra-high vacuum,which may induce artifacts and ultimately reduce one's ability to drawaccurate conclusions from the data. By way of comparison, AFM is aless-destructive high-resolution imaging modality which requires lesssample preparation. Samples imaged using AFM can remain intact, implyingthat measured properties are characteristics of the sample and lesslikely artifacts of the processing or imaging. Other investigators haveutilized AFM to image the collagen matrix in mineralized dentin and boneyielding high-resolution images. However, previous studies did notperform quantitative analyses of the bone ultrastructure to increase theunderstanding of the tissue morphology at this fundamental level oforganization. By using AFM, the morphology of collagen fibrils in fullyintact and mineralized bone can be imaged and quantitatively analyzed tolearn more about the normal nanoscale properties of this material.

The present technology further demonstrates that collagen, the mostabundant protein in animals, exists as a distribution of nanoscalemorphologies in teeth, bones, and tendons. This fundamentalcharacteristic of Type I collagen has not previously been reported andprovides a new understanding of the nanoscale architecture of thisubiquitous and important biological nanomaterial. Dentin, bone, andtendon tissue samples were chosen for their differences in cellularorigin and function, as well as to compare mineralized tissues with atissue that lacks mineral in a normal physiological setting. Adistribution of morphologies was present in all three tissues,confirming that this characteristic is fundamental to Type I collagenregardless of the presence of mineral, cellular origin of the collagen(osteoblast versus odontoblast versus fibroblast), anatomical location,or mechanical function of the tissue.

Bone also has a complex hierarchical structure that has evolved to servestructural and metabolic roles in the body. Due to the complexity of thebone structure and the number of diseases which affect theultrastructural constituents of bone, it is important to developquantitative methods to assess bone nanoscale properties in normal anddisease states. For example, autosomal dominant osteogenesis imperfectaresults predominantly from glycine substitutions (80%) and splice sitemutations (20%) in the genes encoding the α1 or α2 chains of Type Icollagen. Genotype-phenotype correlations using over 830 collagenmutations have revealed that lethal mutations are located in regionscrucial for collagen-ligand binding in the matrix. However, thesecorrelations have not been extended to collagen structure in bone.

The present technology uses an atomic force microscopy-based approach toimage and quantitatively analyze the morphology of Type I collagenfibrils in femora from heterozygous (Brt1/+) mice (α1(I)G349C), comparedto wild type (WT) littermates. This disease system has a well-definedchange in the col1α1 allele, leading to a well characterized alterationin collagen protein structure, which was directly related to alteredType I collagen nanoscale morphology. In Brt1/+ bone, the D-periodicgap/overlap spacing shows significantly greater variability on averageand along the length of the bone compared to WT, although the averagespacing was unchanged. Brt1/+ bone also had a significant difference inthe population distribution of collagen fibrils. These changes may bedue to the mutant collagen structure, or to the heterogeneity ofcollagen monomers in the Brt1/+ matrix. These observations at thenanoscale level provide insight into the structural basis for changespresent in bone composition, geometry and mechanical integrity in Brt1/+bones.

The present technology further demonstrates that there are nosubstantial differences in the nanoscale morphology of collagen betweenmales and females. Since gender is known to play a critical role inregulating properties of the skeleton (composition, size/shape,mechanical integrity), gender-specific differences in the nanoscalemorphology of Type I collagen in bone were investigated. Cortical bonefrom skeletally mature male and female mice was analyzed using an atomicforce microscopy method, testing the hypothesis that differences wouldexist in the mean D-periodic axial gap/overlap spacing and populationdistributions of fibril morphologies as a function of gender. Resultsindicated that the average D-periodic gap/overlap spacing was notsignificantly different in male versus female bones, within the abilityof the methods to detect a difference. Furthermore, there were nodifferences in distributions of D-periodic spacing values. Thesenanoscale observations suggest that although differences exist in thecells derived from male and female animals, gender-specific differencesin bone size, shape and mechanical integrity are likely derived athigher organizational levels of the bone tissue hierarchy.

The following experiments and technical details illustrate aspects ofmethods, systems, apparatus, and compositions relating to thedetermination of whether a subject has a bone disease, whether thesubject is at risk for developing a bone disease, and to screening asubject to determine whether they have a bone disease. In particular,the present technology can determine the distribution of Type I collagenmorphologies in bone and the relation to estrogen depletion. As anexperimental model, bone samples from sham-operated andestrogen-depleted animals were analyzed using the present AFM methods inorder to analyze the collagen fibril morphology of normal bone. Thesemethods could distinguish between healthy bone and bone from animalswith a known disease state. As a result, the present methods increasethe understanding of bone disease mechanisms and provide a complementaryin vitro diagnostic technique for earlier disease detection.

The following materials and methods were employed. Regarding animals,mice from the C3H/He background strain were used with prior approval(University of Michigan, UCUCA protocol #8518). Five male mice weresacrificed at 11 weeks of age by CO₂ inhalation. The right femur fromeach mouse was harvested and the proximal (above the third trochanter)and distal ends were removed using a low-speed sectioning saw (South BayTechnology, Model 650; San Clemente, Calif.) with a diamond waferingblade (Mager Scientific) leaving approximately 7 mm of compact bone ofthe diaphysis. The marrow cavity of each bone was cleaned using a smalltube brush.

Five-year-old Columbia-Ramboulliet cross sheep were anesthetized andovariectomized (OVX, n=3) or subjected to a sham surgery (n=3) [ColoradoState University, ACUC #03-010A-02]. After 2 years, the ewes weresacrificed with an intravenous overdose of a barbiturate, and 2 beamsapproximately 1.75 mm×1.75 mm×9 mm were removed from the mid-diaphysisof the left radius (Les C M, Spence C A, Vance J L, et al. Determinantsof ovine compact bone viscoelastic properties: Effects of architecture,mineralization, and remodeling. Bone 2004; 35:729-38).

Atomic Force Microscopy (AFM) imaging and analysis included thefollowing aspects. Bones were mounted to a steel disk using a thin layerof cyanoacrylate glue (mouse bones were posterior-side down), A flatpolished surface was created using a 3 μm polycrystalline water-baseddiamond suspension and a 0.05 μm deagglomerated alumina suspension(Buehler LTD; Lake Bluff, Ill.). The bones were sonicated for 15 s toremove polishing residue and debris. To remove extrafibrillar surfacemineral and expose underlying collagen fibrils, each bone wasdemineralized using 0.5 M EDTA at a pH of 8.0 for up to 45 min (mousebones only required 5 min of treatment), then vigorously rinsed withultrapure water and soaked at 4° C. for at least 16 h. EDTA is oftenused in bone research to remove mineral while keeping collagen and cellsintact and viable, as described by Jonsson R, Tarkowski A, Klareskog L.A. Demineralization procedure for immunohistopathological use. EDTAtreatment preserves lymphoid cell surface antigens. J Immunol Methods1986: 88:109-14 and Klein-Nulend J, Burger E H, Semeins C M, Raisz L G,Pilbeam C C. Pulsating fluid flow stimulates prostaglandin release andinducible prostaglandin G/H synthase mRNA expression in primary mousebone cells. J Bone Miner Res 1997; 12:45-51. This slow and “gentle”demineralization technique was used in the current study and allowed usto maintain the integrity of the native collagen structure along withthe in fibrillar mineral; see Balooch M, Habelitz S, Kinney J H,Marshall S J, Marshall G W. Mechanical properties of mineralizedcollagen fibrils as influenced by demineralization. J Struct Biol 2008;162:404-10. Before imaging, the sample was briefly sonicated to removeany surface bound mineral.

Samples were imaged in air using a PicoPlus 5500 AFM (Agilent, formerlyMolecular Imaging). Images were acquired in tapping mode using a siliconcantilever with tip radius<10 nm (VistaProbes T300R, force constant 40N/m, resonance frequency 300 kHz, length 125 μm: nanoScienceInstruments; Phoenix, Ariz.) at line scan rates of 2 Hz or lower at 512lines per frame.

Making absolute x-y distance measurements with AFM has multiplelimitations. Prior to sampling, calibration of the system was performedaccording manufacturer guidelines. A calibration grating with a 10-μmperiod was imaged with the scan size set just under the range of thepiezo (80 μm scan size) at 512 pixels by 512 pixels. This results in apixel size of roughly 160 nm. To overcome limitations imposed by pixelsize, multiple consecutive periods were measured. The measured periodwas defined as the total measured length divided by the number ofperiods. The calibration was adjusted until the measured period waswithin 50 nm of the actual 10 μm period. This calibration method resultsin a maximum error of 0.50%, which is less than the 1% tolerancespecified by the manufacturer. For the imaging of collagen fibrils, scansizes were reduced to 3.5 μm at 512×512 pixels. Because of the effectivelinearity of the piezo, error scales with the reduction of scan sizereducing the 50 nm tolerance to 2.2 nm.

Image analysis included the following aspects. Images were acquired fromat least 3 axial locations in each sample. At each location, 3.5 μm×3.5μm amplitude (error) images were analyzed without leveling toinvestigate the D-periodic axial gap/overlap spacing, chosen as our keymetric of fibril morphology. Ten fibrils from each axial location wereanalyzed (SPIP v4.8.2, Image Metrology; Hørsholm, Denmark). Followingimage capture, a rectangular region of interest (ROI) was chosen alongstraight segments of individual fibrils (FIG. 1). The ROI was drawn toensure that it started and ended at the edge of a gap zone, a methodwhich minimizes edge effects that can degrade resolution. For eachevaluated fibril, a two-dimensional Fast Fourier Transform (2D FFT) wasperformed and the primary peak from the 2D power spectrum was analyzedto determine the value of the D-periodic gap/overlap spacing. Thisprocess decouples the measured D-Period repeat distance from both pixelsize and fibril orientation. A detailed analysis of the uncertaintyassociated with this type of measurement in fibrils from the currentstudy demonstrated that using at least 9 D-period repeats sets theminimum bin size for population studies at 0.8 nm. For all histograms,the bin size was therefore set at 1 nm.

Statistical analysis included the following aspects. All statisticalanalyses utilized SPSS (Version 16.0. SPSS Inc.). For allinvestigations, a value of p<0.05 was considered significant. Toinvestigate differences in fibril morphology between species and due toestrogen deficiency, D-periodic axial spacing values measured from anindividual sample were pooled, yielding an average value for thatsample. The values from mice (n=5), sham sheep (n=6) and OVX sheep (n=5)were then compared using one-way ANOVA with post hoc Bonferroni tests.

To examine differences in the distribution of fibril morphology betweensham and OVX groups, the Cumulative Distribution Function (CDF) of eachgroup was computed. The CDF shows what fraction of a given sample iscontained up to a particular value, easily demonstrating differencesbetween distributions in both mean and standard deviation. In FIG. 5 b,Cumulative Group Total (%) is used on the y-axis to make it clear thatthe CDF is a percentage within each group up to each spacing value. Totest for statistical significance between distributions,Kolmogorov-Smirnov (K-S) tests were then applied to the data. This testis sensitive to changes in both the mean value and standard deviation ofa distribution.

The following results were obtained. To quantitatively assess the axialgap/overlap spacing in murine cortical bone as a baseline, femora from11-week-old male mice of the C3H/He background strain were used. In theintracortical bone along the anterior surface of the diaphysis, 3 axiallocations were analyzed. At each axial location, a 10 μm×10 μm scan wasperformed to find suitable sites for closer inspection. FIG. 2 shows arepresentative 3D rendering of a bone surface displaying bundles ofwell-oriented fibrils surrounded by less organized fibrils in a wovenconfiguration. At least 10 fibrils were chosen in each axial location,yielding at least 30 measurements from each bone. In total, 193 fibrilswere analyzed.

For each of the analyzed bones, the measurements within each bone werepooled to yield the mean fibril spacing for that bone. These mean valuesranged from 67.7 nm to 69.0 nm (FIG. 3 a), with an overall mean axialspacing for all 5 bones of 68.3±2.4 nm. The median value for all 193measures of 68.2 nm was in good agreement with the mean. The dashedhorizontal lines in FIGS. 3 a and 4 are for reference and correspond tothe 67 nm axial spacing that is predicted by the Hodge-Petruska model(Hodge A J, Petruska J A. Recent studies with the electron microscope onordered aggregates of the tropocollagen molecule. In: Ramachandran G N,editor. Aspects of protein structure. New York; Academic Press; 1963. p.289). As noted in the materials and methods, the ability to measureabsolute distances with AFM was limited by the about 2.2 nm toleranceset during the calibration of our system. Therefore, it is not possibleto state that there is an absolute difference between the 68.3 nm meanin the mouse fibrils and the 67 nm value predicted by Hodge andPetruska. The histogram of all 193 fibrils shows that there is adistribution of spacings ranging from 63 nm to 74 nm (FIG. 3 b).

Using a model of estrogen-depletion in sheep, it was hypothesized thatit would be possible to distinguish between normal bone and bone fromanimals with a known disease state by monitoring changes in thedistribution of collagen fibril morphologies. Samples fromovariectomized (OVX) and sham-operated sheep were processed as describedin the materials and methods. It was found that the fibrils from thesham sheep were similar in terms of mean (68.0±2.6 nm) and distributionwhen statistically compared with mouse fibrils (FIG. 4, p=1.00 forANOVA, p=0.319 for KS test). However, comparison of sham samples (n=6)to OVX samples (n=5) showed striking differences. The mean from the OVXsamples was 65.9±3.1 nm. When the mean values from Sham and OVX sampleswere compared by one-way ANOVA, there was a significant effect ofestrogen-depletion on the fibril spacing (p=0.048), When the fibrils ineach group are viewed in histogram form, there are different populationsof fibrils within the 2 groups (FIG. 5). The bins which contain the mainpopulation of fibrils in each group were selected such that they containthe mean of the sham fibrils ±1 standard deviation (65 nm to 71 nm). Thesham bones had a population of fibrils (11.5% of total fibril measured)with spacings of 72 nm or greater which was almost completely absentfrom the OVX group (1.8%). Similarly, the OVX bones had a population offibrils with spacings of 64 nm or less (28.0%) which was less prominentin the sham bones (7.1%). When compared using a K-S test, the CDFs fromthe groups was significantly different (p<0.001) indicating asignificant difference in the population distributions of the groups(FIG. 5 b).

Our data indicate that as opposed to a single value, normal bonecontains a distribution of collagen fibril D-periodic axial gap/overlapspacings. This measure captures many aspects of the fibril hierarchy andcan be related to the state of the individual triple helices,posttranslational modifications and cross-linking. By monitoring thisdistribution in normal bone and bone from estrogen-depleted animals, wehave demonstrated the ability to detect disease-induced differences inthe Type I collagen ultrastructure of bone.

In this study, 2D FFTs were used to determine the value of theD-periodic gap/overlap spacing (FIG. 1). At least one other study hasused analysis by FFT to derive spacing data from AFM images of collagen(Habelitz S, Balooch M, Marshall S J, Balooch G, Marshall Jr G W, Insitu atomic force microscopy of partially demineralized human dentincollagen fibrils, J Struct Biol 2002; 138:227-36). This study relied onthe 1D FFT, a method which allows the user to determine the powerspectrum of the wavelength component along a single line in an image.This method is dependent on the angular orientation of a line that isdrawn on the collagen fibril by the user and can lead to errors in themeasured spacing values. From our calculation, as little as a 5°deviation in the line that is drawn from the normal direction of thefibril spacing can alter the measured spacing by as much as 5% to 10%,depending on the measured length of the fibril. A 10% error on ameasurement of a 67-nm fibril means that the spacing value could rangefrom 60-74 nm, making it impossible to detect the type of changes infibril morphology seen and discussed herein. The combination of moreaccurate measures and the assessment of variability associated withthose measures provide us with confidence that the values we obtain aretrue characteristics of the tissues being measured.

To quantitatively assess the Type I collagen morphology in murinecompact bone as a baseline, femora from 11-week-old male mice from theC3H/He background strain were used. The use of a specific age, gender,and background strain of mouse guarantees that the known influence ofthese properties on skeletal structure and function were avoided.Further, by investigating multiple axial locations along the length ofone type of bone, the effect of variations within and between bones wasreduced. FIG. 2 shows a representative 3D rendering of a bone surfacedisplaying bundles of well-oriented fibrils surrounded by less organizedfibrils in a woven configuration.

Using 2D FFT analyses, we found a distribution of spacings in normalmurine bone ranging from 63 nm to 74 nm (FIG. 3). Although the mostcommonly accepted value for this D-periodic spacing is 67 nm (aspredicted by Hodge and Petruska), this value is based on a theoreticalmodel of a single collagen fibril in isolation. As mentioned above, theability to accurately measure absolute distances using AFM isintrinsically limited by the calibration process. The error associatedwith these absolute measurements therefore reduces our ability to make acomparison of the absolute mean to the expected 67 nm value, as thedifference between these values is within the error of the technique.However, the strength of this study is that a distribution of spacingvalues was present in normal tissue. This range of values is a realobservation and is not impacted by the absolute error. The concept of adistribution is often overlooked in measurements of collagen, and themean value is reported without explanation.

A study in dentin, for example, showed a distribution in fibrilspacings, but reported that there was a “relatively narrow distribution”in the measurements around 67 nm (Habelitz S, Balooch M, Marshall S J,Balooch G, Marshall Jr G W. In situ atomic force microscopy of partiallydemineralized human dentin collagen fibrils. J Struct Biol 2002;138:227-36). We hypothesize that although the mean value of theD-periodic spacing in a mineralized fibril is important, thedistribution itself may have even greater significance. Thisdistribution of fibril morphologies is found in wild type bone meaningthat it is a fundamental property of normal tissue. The influences thata distribution of collagen morphologies (versus a single value) couldhave on the mechanical properties of the tissue are profound. Bone is aheterogeneous tissue with anisotropic mechanical properties. Given thata whole bone is typically under a complex loading state includingtension, compression and shear, it is imperative that the structure as awhole be mechanically competent in multiple directions. The materialitself is much more specialized and the anisotropic tissue propertiesthat contribute to overall mechanical competence begin at the mostfundamental levels of the tissue hierarchy. It is likely that thedistribution of fibril morphologies measured here is partiallyresponsible.

Alterations in the distribution of fibril morphology in cases of diseasemay indicate flaws in the collagen fibrils from misalignment orover-modification of the individual collagen molecules within thefibrils, changes that could be compensated for over larger lengthscales. Using a model of estrogen-depletion in sheep, it washypothesized that it would be possible to distinguish between normalbone and bone from animals with a known disease state by monitoringchanges in this distribution. The fibrils from the sham sheep weresimilar in terms of mean and distribution compared with the normal mousefibrils (FIG. 4), verifying the utility of this method in differentspecies. However, a comparison of sham sheep to OVX sheep showedstriking differences both in terms of overall mean as well as indistribution (FIGS. 4 and 5). This ability to measure fibril morphologyand to distinguish between normal and diseased bone not only provides apowerful method to study the mechanism of disease at the nanoscale, butit also has important implications to the diagnosis of disease in boneand other collagenous tissues (e.g., dentin, tendon, skin, ligament).

The current gold-standard for diagnosis of osteoporosis focusesexclusively on BMD, as measured from a 2D DEXA projection, and istherefore dependent on bone size and shape. Further, using BMD todiagnose osteoporosis is problematic, as normal BMD does not guaranteeprotection from fracture and many osteoporosis cases do not becomeclinically evident until fracture occurs. The current study shows thatmeasuring properties of the organic matrix may provide powerfulcomplementary information to BMD, as changes in collagen may becomeapparent earlier in the progression of the disease. Although this methodwould currently require a biopsy, something that is a standard practicefor the diagnosis of many bone diseases, the benefit of earlierdiagnosis and treatment may outweigh the risk.

Although AFM requires less sample preparation than other high resolutionimaging modalities, there is still the possibility of artifacts forsample preparation and imaging. Samples in the current study were imagedin air meaning that contraction of fibrils could occur from dehydration.However, most examples of contraction were observed using electronmicroscopy which fully dehydrates the tissue and requires ultra-highvacuum in our experience imaging bone, dentin, and tendon from multiplespecies and from normal and diseased animals, contraction of fibrilspacing or the appearance of new fibril populations due to dehydrationhas never been observed.

The collagen in bone is impregnated with and surrounded by mineral whichmust be removed. After 30 min of continuous treatment with 10% citricacid, intrafibrillar mineral is protected from demineralization.Further, treatment with EDTA removes extrafibrilla mineral while leavingthe underlying collagen intact (Kindt J H, Thurner P J, Lauer M E, etal. In situ observation of fluoride-ion-induced hydroxyapatite-collagendetachment on bone fracture surfaces by atomic force microscopy.Nanotechnology 2007; 135102:18). EDTA can remove mineral while keepingcells and collagen intact and viable. Mouse bones were only treated forabout 5 min with EDTA while the sheep bones required up to 45 min. Therewere no differences in either mean spacing value or in the distributionof fibril morphologies between the mouse bones and the sham sheep bones.Further the comparison between bone, dentin (also mineralized) and tailtendon (not mineralized and, therefore, not treated with EDTA) showed nodifferences in mean fibril spacing. This comparison between sampleswhich required sonication (bones and teeth) and samples which did not(tendons) further indicates that the effects of sonication were minimal,All samples in the current study were imaged under identical conditionsand we are interested only in relative differences between groups.Therefore the differences seen here are likely true disease-inducedchanges in Type I collagen morphology.

In sum, the current technology demonstrates the ability to utilize AFMimaging to make quantitative assessments of the distribution of collagenfibril morphologies in bone. It is clearly shown that normal bonecontains a distribution of collagen fibril morphologies and that changesin this distribution can be directly related to disease state.Osteoporosis is a significant medical and economic burden facing oursociety and is projected to worsen with the aging population. Althoughmore research on the causes of this disease is important, earlydiagnosis and treatment is imperative. Monitoring changes in the fibrilmorphology distribution as a function of disease not only increases ourfundamental understanding of bone as a material, but also providesimportant insight into disease progression and offers the possibility ofa much-needed in vitro procedure to complement the use of BMD fordisease diagnosis.

The present technology further demonstrates that Type I collagen existsas a distribution of nanoscale morphologies in teeth, bones, andtendons. Collagens are the most abundant structural proteins in animals.In humans, there are currently 28 proteins known as collagens serving avariety of structural roles including shaping and organizingextracellular matrices (ECM), providing a scaffolding for tissueformation, cell adhesion and migration, as well serving as the principalsource of tensile strength in animal tissues. The hallmarkcharacteristic of collagen is a macromolecule composed of trimericpolypeptide chains, each comprising regions including a repeatingGly-X-Y triplet (X and Y are often proline and hydroxyproline,respectively). One major classification of collagen is thefibrillar-forming type, which has an approximately 300 nm long,uninterrupted triple helix (with the exceptions of Type XXIV and XXVII).

Type I collagen is archetypal in that it is trimeric, possesses anuninterrupted Gly-X-Y triple helix, and assembles into structuralfibrils. With the exception of cartilaginous tissues, Type I collagen isfound throughout the body in tissues such as teeth, bones, tendons,skin, arterial walls, and the cornea. Type I collagen is alsosynthesized in response to injury and is present in scar tissue. On thebasis of the seminal work of Hodge and Petruska in 1963 (and earlierwork by many others), the primary morphological characteristic of Type Icollagen fibrils, the D-periodic axial gap/overlap spacing, was shown tobe 67 nm based on theoretical models of a single collagen fibril inisolation. In the 47 years since this assertion, X-ray diffraction andelectron microscopy studies have supported this singular spacing valuewithin the error of the individual technique. Given the complexity ofthe collagen fibril itself, the range of tissues in which these fibrilsare incorporated, and the potential for morphology variation withdisease, it seems unlikely that a single spacing value would exist forall fibrils. A recent study using atomic force microscopy (AFM) noted adistribution of fibril spacing values in dentin, but did notstatistically analyze the distribution or discuss its biological ormechanical significance (Habelitz, S.; Balooch, M.; Marshall, S. J.;Balooch, G.; Marshall, G. W., Jr. J. Struct. Biol. 2002, 138, 227-236).

Measuring and understanding morphological features of the ultrastructureof collagen in collagen-based tissues is imperative to our understandingof normal tissue architecture. The need for accurate quantitativeanalytical methods to assess collagen's nanoscale morphology with aslittle disruption to the tissue as possible has prompted us to study thecollagen ultrastructure of various tissues using AFM. As opposed toother methods which are used to image the Type I collagen ultrastructureof tissues, samples imaged using AFM remain intact to a greater degree,increasing confidence that measured properties are samplecharacteristics rather than artifacts of processing or imaging. AlthoughAFM has been used to image the collagen matrix in mineralized dentin andbone, and other nonmineralized tissues, the current study uses aquantitative and statistically powered technique to analyze thenanoscale morphology of individual Type I collagen fibrils in multipletissues from the same animal.

In the current study, it was hypothesized that AFM could be used toimage and quantitatively analyze the morphology of Type I collagenfibrils in fully intact and mineralized teeth and bones, as well as innonmineralized tendons, to learn more about the normal nanoscaleproperties of these materials. These three tissues were chosen based ontheir differences in cellular origin and function. Further, sincetendons lack mineral under normal physiological conditions, tendonsamples served as an internal control to verify that thedemineralization process used on bone and dentin samples did not disruptthe native collagen structure of these tissues within the detectionlimits of the AFM methodology.

The following materials and methods were employed. With respect toanimals, eight-week-old male mice from the mixed Sv129/CD-1/C57BL/6Sbackground strain were used with prior approval (University of Michigan,UCUCA protocol #09637). After sacrifice by CO₂ inhalation, femora,mandibular incisors, and tails were harvested, wrapped in gauze soakedwith calcium-buffered saline, and stored at −20° C.

Atomic Force Microscopy (AFM) imaging and analysis included thefollowing aspects. Before use, the proximal (above the third trochanter)and distal ends of each left femur were removed using a low-speedsectioning saw (South Bay Technology, model 650; San Clemente, Calif.)with a diamond wafering blade (Mager Scientific) leaving approximately 7mm of compact bone of the diaphysis (FIG. 1). The marrow cavity of eachbone was cleaned using a small tube brush.

Mandibular incisors were cleaned of soft tissue and surrounding bone.Bones (anterior side facing up) and teeth (lateral side facing up) weremounted onto a steel disk using a thin layer of cyanoacrylate glue. Aflat polished surface was created using a 3 μm polycrystallinewater-based diamond suspension and a 0.05 μm deagglomerated aluminasuspension (Buehler LTD; Lake Bluff, Ill.). This polishing exposedintracortical bone in the bone samples and dentin in the teeth samples.The bones and teeth were sonicated for 15 s to remove polishing residueand debris. To remove extrafibrillar surface mineral and exposeunderlying collagen fibrils, each bone and tooth was demineralized using0.5 M EDTA at a pH of 8.0 for up to 15 min, then vigorously rinsed withultrapure water and soaked at 4° C. for at least 16 h. EDTA is oftenused in mineralized tissue research to remove mineral while keepingcollagen and cells intact and viable. This demineralization technique isslower than other treatments and allowed us to maintain the integrity ofthe native collagen structure along with the intrafibrillar mineral.Before imaging, each sample was briefly sonicated to remove any mineralthat was still bound to the surface.

To isolate tendons from each tail, the tip of the tail was removed usinga scalpel. The skin was peeled back at the base of the tail and removedin a base-to-tip direction, exposing the underlying tendons. Tendonswere removed whole, minced using a scalpel and scissors, and thenhomogenized in 1-2 mL of ultrapure water to disrupt the fasciclestructure and release collagen fibrils. Fibril containing solutions weredeposited onto freshly cleaved mica surfaces and allowed to dry at roomtemperature.

Samples were imaged in air using a PicoPlus 5500 AFM (Agilent). Dentinand bone were imaged in tapping mode using silicon cantilevers(VistaProbes T300R, tip radius<10 nm, force constant 40 N/m, resonancefrequency 300 kHz; nanoScience Instruments; Phoenix, Ariz.). Tendonfibrils were imaged in contact mode using silicon nitride cantilevers(Veeco DNP, tip radius about 20 nm, force constant 0.58 N/m). Imageswere acquired from 3 locations in each of 5 teeth, 9 locations in eachof 4 bones (designated 1-9 beginning at the proximal end of the sample,FIG. 6), and several locations in each of 4 tendon samples. At eachlocation, 3.5 μm×3.5 μm amplitude or deflection images were analyzed toinvestigate the D-periodic spacing. In teeth, 15-20 fibrils wereanalyzed in each location. In bones, 5-10 fibrils were analyzed in eachlocation (with the exception of 1 bone which lost locations 8 and 9during polishing). In each tendon sample, 35-50 fibrils were analyzed.Representative images and line scans from each tissue type are shown inFIG. 7.

Image Analysis included the following aspects. After image capture, arectangular region of interest (ROI) was chosen along straight segmentsof individual fibrils (FIG. 3 a; SPIP v 4.8.2, Image Metrology;Hørsholm, Denmark, FIG. 7). The ROI was drawn to ensure that it startedand ended at the edge of a gap zone, which minimizes edge effects thatcan degrade resolution. For each evaluated fibril, a two-dimensionalfast Fourier transform 2D FFT) was performed and the primary peak fromthe 2D power spectrum was used to determine the value of the D-periodicgap/overlap spacing (FIG. 8 b). This process decouples the measuredD-Period repeat distance from both pixel size and fibril orientation. Intotal, 291 dentin fibrils, 288 bone fibrils, and 160 tendon fibrils wereanalyzed.

Making absolute x-y distance measurements with AFM can have limitations.Prior to sampling, calibration of the system was performed accordingmanufacturer guidelines. A calibration grating with a 10 μm period wasimaged with the scan size set just under the range of the piezo (80 μmscan size) at 512 pixels by 512 pixels. This results in a pixel size ofroughly 160 nm. To overcome limitations imposed by pixel size, multipleconsecutive periods were measured. The measured period was defined asthe total measured length divided by the number of periods. Thecalibration was adjusted until the measured period was within 25 nm ofthe actual 10 μm period. This calibration method results in a maximumerror of 0.25%, which is less than the 1% tolerance specified by themanufacturer. For the imaging of collagen fibrils, scan sizes werereduced to 3.5 μm at 512×512 pixels. Because of the effective linearityof the piezo, error scales with the reduction of scan size reducing the25 nm tolerance to 1.1 nm.

Statistical Analysis included the following aspects. All statisticalanalyses utilized SPSS (version 16.0, SPSS Inc.). For allinvestigations, a value of p<0.05 was considered significant. Toinvestigate differences in fibril morphology as a function of tissuetype, D-periodic axial spacing values measured from an individual samplewere pooled, yielding an average value for that sample. The values fromdentin (n=5), bone (n=4), and tendon (n=4) were then compared by one wayANOVA with posthoc Bonferroni tests.

To examine differences in the distribution of fibril morphology amongtissue types, histograms and the cumulative distribution function (CDF)of each group were computed. The CDF shows what fraction of a givensample is contained up to a particular value, easily demonstratingdifferences among distributions in both mean and standard deviation. InFIG. 11 b, cumulative total (%) is used on the y-axis to make it clearthat the CDF is a percentage within each group up to each spacing value.To test for statistical significance between distributions,Kolmogorov-Smirnov (K-S) tests were then applied to the data. This testis sensitive to changes in both the mean value and standard deviation ofa distribution.

The D-periodic axial gap/overlap spacing was chosen as the key metric offibril morphology. This measure captures aspects of fibril structurewhich may be related to the state of the individual molecular triplehelices, post-translational modifications, and cross-linking. The mostcommonly accepted value for the D-periodic spacing of Type I collagen is67 nm (as predicted by Hodge and Petruska), but this value is based on atheoretical model of a single collagen fibril in isolation. Themolecular origin of this morphological feature has not been completelyelucidated. A recent study performed AFM and TEM analyses on identicalcollagen fibrils (Lin, A. C.; Goh, M. C. Proteins Struct., Funct.,Genet. 2002, 49, 378-384). The paper suggested that the tallerprotrusions seen on the surface of collagen fibrils using AFM registerperfectly with the dark bands seen in TEM, suggesting that there is morematerial in these bumps and that this material is better at scatteringelectrons. As mentioned by the authors, this observation does not ruleout the existence of gap zones as proposed by Hodge and Petruska.Regardless of the origin of the D-periodic spacing, this morphologicalfeature is well-resolved with AFM and easy to quantify.

To quantitatively assess the D-periodic spacing in Type I collagen-basedtissues as a function of tissue type, mandibular incisors, femora, andtails were harvested from 8-week-old male mice. The use of a specificage, gender, and background strain of mouse guarantees that the knowninfluence of these properties on skeletal structure and function wasavoided.

Along the length of each bone, measurements were made at nine axiallocations (FIG. 6). By pooling data from the four bones at each of thenine locations and analyzing the D-periodic spacing, it was confirmedthat there were no systemic changes in Type I collagen morphology as afunction of axial location in the bone (FIG. 9). The boxes represent themiddle 50% of the data, and the whiskers depict the data extremes. Thediamond is the mean, and the line within the box is the median of eachgroup. The dashed horizontal line is for reference and corresponds tothe 67 nm axial spacing that is predicted by the Hodge-Petruska model.This type of data presentation is only possible for the bone samples.Dentin samples were measured from three locations in each tooth.However, because of differences in tooth geometry, these locations werenot identical from tooth to tooth. Further, the tendon samples wereadsorbed on mica so anatomical location is not applicable.

Measurements within each tooth, bone, and tail sample were pooled toyield the mean fibril spacing for that sample. Tooth (n=5), bone (n=4),and tendon samples (n=4) were then compared; the overall mean valueswithin each tissue type were 67.8, 67.3, and 67.9 nm for dentin, bone,and tendon, respectively (FIG. 10). On the basis of system calibration,the ability to measure absolute distances has an error of approximately1.1 nm. Therefore, it is not possible to state that the measured meansin this study are different from the 67 nm value predicted by Hodge andPetruska. When the mean values in each group were compared by one wayANOVA with posthoc Bonferroni tests, no differences were present betweenany of the groups (FIG. 10B; a p-value of <0.05 was consideredsignificant).

FIG. 11 shows that a distribution of fibril morphologies existed in eachgroup. The concept of a distribution is often overlooked in measurementsof collagen, and the mean value for the D-periodic spacing is sometimesreported without explanation. The main population of fibrils in eachgroup was selected such that the bins contained the mean of the bonefibrils (±1 standard deviation (66 to 69 nm, FIG. 11 a). Within thisrange, 75% of bone fibrils were found in comparison to 85% for dentinand 60% for tendon fibrils. As a second method to visualize thedistributions, the cumulative distribution function (CDF) was computedfrom the measurements in each group (FIG. 11 b). To test for statisticalsignificance between distributions, Kolmogorov-Smirnov (K-S) tests wereapplied to the data. This test is sensitive to changes in both the meanvalue and standard deviation of a distribution. The distributions fromthe three tissue types were statistically distinguishable from oneanother (FIG. 11 b: dentin vs. bone, p<0.001; dentin vs. tendon,p=0.004; bone vs. tendon; p<0.001). Despite these differences, thepresence of a distribution was a common feature in all three tissues,indicating that a distribution of fibril morphologies is a definingnanoscale characteristic of Type I collagen.

The presence of a distribution in fibrils is interesting and important,as this distribution may hold the key to understanding important aspectsof the ultrastructure of many collagen-based tissues. Explaining thereasons behind the presence of a distribution in fibril morphologies isnot trivial. Because of the structural hierarchy of collagen fibrils, adistribution in the D-periodic spacing near the ideal value predicted byHodge and Petruska is most likely driven by alterations in the end-toend spacing of collagen molecules within the fibril, or by a change inthe tightness of the twist of the fibril. Either of these mechanismscould cause the measured D-period spacing at the fibrillar-level tochange. Another possibility is the presence of proteins both within andon the surface of collagen fibrils, which may lead to changes in theobserved spacings. Another potential source of variation is the presenceof intramolecular and intermolecular crosslinks, which could not onlyadd heterogeneity to the structure, but also allow the spacing tofurther change as the crosslinks mature.

One drawback when working with mineralized tissues like bone and dentinis that, in order to analyze the organic component by AFM, some portionof the inorganic mineral must be removed to expose the underlyingstructure. Regardless of the method used, there is the possibility thatthis treatment could have important effects on the properties that onehopes to measure. Many methods are used in the literature to remove thismineral for imaging studies such as AFM. A majority of studies use someform of acid which etches mineral from the surface to expose collagen.In our hands, these methods worked rapidly and often completelydestroyed the surface (e.g., craterlike hole would appear on the surfacewithin 1-2 min, but no defined collagen fibrils were visible). Incontrast to these acid treatments, EDTA is a chelating agent which isslow at removing calcium from bone and is close to physiological pH.With long-term treatment (days to weeks), EDTA can fully demineralizebone and dentin. In fact, EDTA is often used to remove mineral andliberate trapped osteocytes for cell culture experiments. In the currentstudy, every effort was made to treat the samples for as little time aspossible to expose the underlying collagen fibrils.

It is important to keep in mind that, although reports vary, roughly ⅓of the mineral present in the extracellular matrix (ECM) of mineralizedtissues is intrafibrillar (i.e., present within the collagen fibrilsthemselves). This fact suggests that short periods of EDTA treatment arelikely to preferentially remove extrafibrillar mineral first, leavingbehind the intrafibrillar mineral which helps to stabilize the structureof the ECM. Two recent AFM studies directly assess the effects of citricacid (Balooch, M.; Habelitz, S.; Kinney, J. H.; Marshall, S. J.;Marshall, G. W. J. Struct. Biol. 2008, 162, 404-410.) or EDTA (Kindt, J.H.; Thurner, P. J.; Lauer, M. E.; Bosma, B. L.; Schitter, G.; Fantner,G. E.; Izumi, M.; Weaver, J. C.; Morse, D. E.; Hansma, P. K.Nanotechnology 2007, 18, 135102) on mineralized collagen fibrils. Thefirst paper follows the effects of citric acid over time and found thatintrafibrillar mineral is protected from demineralization, even after 30min of continuous treatment. The second study showed that treatment withboth NaF and EDTA removed extrafibrillar mineral while leaving theunderlying collagen structure intact. In the current study,nonmineralized tendon samples served as an internal control to verifythat the demineralization process used on bone and dentin samples didnot alter collagen structure within the ability of our AFM technique todetect changes in morphology. The comparison of tendon to bone anddentin also verified that the presence of mineral is not the source ofthe observed distributions in bone and dentin.

The tissues analyzed in the current study serve a variety of mechanicalroles. Dentin is less mineralized and less brittle than the enamel thatcovers it and primarily supports compressive loads from mastication(dentin in the mouse mandibular incisor may also be subject to tensileloads given its shape). Tendons provide a link between bones and musclesand are subject to tensile loading. Bone is a heterogeneous tissue, anddepending on location, bone can be subjected to a highly complex loadingstate including tension, compression, and torsion. Despite thesedifferences in mechanical need, all three tissues displayed a similardistribution of collagen fibril morphologies. The influences that such adistribution may have on the mechanical integrity of a tissue areunknown. However, it is possible that, as the spacing in a fibril getsshorter, less space is available in the fibril for the presence of wateror mature crosslinks. This could detrimentally impact the viscoelasticand post-yield properties of the tissue, while possibly leading to astiffer fibril. The impacts that such changes have on thestructural-level properties of a tissue are complicated given thehierarchical complexity of collagen-containing tissues.

Given the ubiquitous nature of Type I collagen in animals, and thenumber of disease that can affect collagen-containing tissues, it seemsprobable that alterations in the Type I collagen ultrastructure willoccur with disease. In fact, the present technology shows that forsamples from normal and estrogen-depleted sheep (all of which weretreated and imaged in exactly the same way), not only is the mean fibrilspacing significantly changed, but so too is the distribution of fibrilmorphologies. The fact that disease-induced changes in the distributionof fibril morphologies were observed further indicates that the presenceof a distribution is not driven by artifact. Age-dependent diseases suchas estrogen-depletion-related osteoporosis, which is not correlated withgenetic alteration, can lead to collagen pathologies which aredetectable using AFM. Therefore, monitoring this distribution representsa new diagnostic technique for disease in a number of collagen-basedtissues.

Some concern may be raised over the imaging conditions used in thecurrent study. Dehydration of collagen fibrils has been observed tochange the normal d-periodic spacing of the fibrils. The majority ofthese observations were made using electron microscopy, a method thatnot only fully dehydrates the tissue, but also requires ultrahighvacuum. When using AFM in the current technology (a method that does notrequire ultrahigh vacuum), tissues were processed and stored wet. Justbefore imaging, the surface of the bones and teeth were briefly blowndry with nitrogen and imaged immediately under atmospheric pressure. Thetendon samples are allowed to dry in air for around 1 h, and then imagedunder the same conditions. It is likely that the sample was not fullydehydrated even after the entire sample had been imaged. Regardless, allsamples in the current technology were imaged under identicalconditions, and only relative differences between tissue types wereanalyzed.

In sum, the present technology demonstrates that, regardless of cellularorigin or anatomical location, normal Type I collagen-based tissuescontain a distribution of nanoscale collagen morphologies as measuredusing the D-periodic gap/overlap spacing. The mean differences in theD-period spacing are not statistically significant; however, thedifferences in the distributions of the D-period spacing as a functionof tissue type are statistically significant. This is a new andimportant observation on the nanoscale ultrastructure of the mostabundant protein in animals. The presence of this distribution may holdpowerful information about the ultrastructure of collagen-based tissues.Further, monitoring this distribution represents a new diagnostictechnique for disease in a number of collagen based-tissues.

The present technology further demonstrates that the nanoscalemorphology of type I collagen is altered in the Brt1 mouse model ofOsteogenesis Imperfecta (OI). Type I collagen forms the scaffolding uponwhich all bones are built. Mutations in the genes encoding the α chainswhich form the triple helical collagen molecule can cause diseases ofbone and other tissues such as OI, or brittle bone disease, and someforms of Ehlers-Danlos Syndrome. Type I collagen is a heterotrimericmolecule composed of two α1 chains and one α2 chain. Human OI resultsprimarily from point mutations that cause the substitution of a triplehelical glycine residue (80%) or splice site mutations (20%) in thegenes encoding the α1 or α2 chains of Type I collagen. The diseasedisplays a wide spectrum of clinical severities, with more severe formsarising from changes in the primary amino acid sequence. These changesin alpha chain structure result in delayed protein folding andover-modification of the collagen triple helix, and decrease collagenquality, although disease severity does not correlate with the extent ofcollagen over-modification. Milder forms of dominant OI arise from nullmutations in one Type I collagen allele, resulting in an underproductionof normal collagen. Depending on the severity of the disease, symptomsrange from susceptibility to fracture from mild trauma to perinatallethality.

Correlations between genotype and phenotype in OI utilizing over 800mutations revealed that mutations in the Major Ligand Binding Regions inthe α1 chain and the proteoglycan binding regions in the α2 chain werealmost entirely lethal. Histomorphometry of OI bone has shown thatsurface-based remodeling parameters are increased in all forms of OI butthat mineralization defects are not present (with the possible exceptionof Type VI OI). However, correlations between genotype and collagenstructure in OI bone have not been made. The Brittle (Brt1) mouse, amodel of human Type IV OI which has a classical glycine substitution(G349C) in one col1α1 allele, was created to study how specific changesin the amino acid sequence of collagen can lead to OI phenotypiccharacteristics.

The need for quantitative analytical methods to assess the nanoscalemorphology of collagen in bone without tissue disruption has prompted usto study the collagen ultrastructure of bone using atomic forcemicroscopy (AFM). In comparison to other high-resolution methods used toassess the nanoscale properties of bone and other mineralized tissues,AFM is less destructive and requires less sample preparation, implyingthat measured properties are less likely artifacts of sample processingor imaging. It was previously shown that normal Type I collagen-basedtissues, including bone, contain a distribution of Type I collagenfibril morphologies. We further demonstrated that estrogen-depletion insheep leads to a significant change in this distribution in bone.However, because the effects of estrogen on collagen structure are notwell understood, it was not possible to ascribe a mechanism to thesechanges in collagen morphology. In the experiments presented below, AFMwas used to image and quantitatively analyze the morphology of Type Icollagen fibrils in intact and mineralized bones from wild type (WT) andheterozygous (Brt1/+) mice. This disease system has a well-definedchange in the col1α1 allele and in collagen protein structure, which aredirectly related to altered Type I collagen nanoscale morphology. It washypothesized that changes in the mean D-periodic axial gap/overlapspacing as well as alterations in the population distribution of fibrilmorphologies, might be present in diseased fibrils from Brt1/+ mice incomparison to their WT littermates. Although the average D-periodicgap/overlap spacing was not significantly different in Brt1/+ versus WTbone, the D-periodic gap/overlap spacing in Brt1/+ bone had greatervariability overall and along the length of the bone. A significantdifference in the collagen population distributions was also present inBrt1/+ bone. These nanoscale observations provide insight into thestructural basis for changes present in bone composition, geometry andmechanical integrity in Brt1/+ bones.

Materials and methods employed include the following aspects. Brt1/+ andWT mice from the mixed Sv129/CD-1/C57BL/6S background strain were usedwith prior approval of the NICHD Animal Care Committee (protocol # ASP09-023) and were sacrificed by lethal injection. Femurs from twomonth-old male wild type (WT, n=4) and Brt1/+ (n=4) mice were harvestedand stripped of soft tissue before processing for imaging

Atomic Force Microscopy (AFM) imaging and analysis included thefollowing aspects. Before use, the proximal (above the third trochanter)and distal ends of each right femur were removed using a low-speedsectioning saw (FIG. 6). The marrow cavity of each bone was cleanedusing a small tube brush. Bones were mounted, anterior-side up, to asteel disk using a thin layer of cyanoacrylate glue. A flat polishedsurface of intracortical bone was created using a 3 μm polycrystallinewater-based diamond suspension and a 0.05 μm deagglomerated aluminasuspension (Buehler LTD; Lake Bluff, Ill.). The bones were sonicated for15 seconds to remove polishing residue and debris. To removeextrafibrillar surface mineral and expose underlying collagen fibrils,the surface of each bone was demineralized using 0.5M EDTA at a pH of8.0 for 15 minutes, then vigorously rinsed with ultrapure water andsoaked at 4° C. for at least 16 hours. EDTA is often used in mineralizedtissue research to remove mineral while keeping collagen and cellsintact and viable. Before imaging, each sample was briefly sonicated toremove any mineral that was still bound to the surface.

Samples were imaged in air using a PicoPlus 5500 AFM (Agilent). Imageswere acquired in tapping mode using silicon cantilevers (VistaProbesT300R, tip radius<10 nm, force constant 40 N/m, resonance frequency 300kHz; nanoScience Instruments; Phoenix, Ariz.) at line scan rates of 2 Hzor lower at 512 lines per frame.

Making absolute x-y distance measurements with AFM can presentlimitations. Prior to sampling, calibration of the system was performedaccording manufacturer guidelines. A calibration grating with a 10 μmperiod was imaged with the scan size set just under the range of thepiezo (80 μm scan size) at 512 pixels by 512 pixels. This results in apixel size of roughly 160 nm. To overcome limitations imposed by pixelsize, multiple consecutive periods were measured. The measured periodwas defined as the total measured length divided by the number ofperiods. The calibration was adjusted until the measured period waswithin 25 nm of the actual 10 μm period. This calibration method resultsin a maximum error of 0.25%, which is less than the 1% tolerancespecified by the manufacturer. For the imaging of collagen fibrils, scansizes were reduced to 3.5 μm at 512×512 pixels. Because of the effectivelinearity of the piezo, error scales with the reduction of scan size,reducing the 25 nm tolerance to 1.1 nm.

Images were acquired from 9 axial locations in each bone sample(designated 1-9 beginning at the proximal end of the sample, FIG. 6). Ateach location, 3.5 μm×3.5 μm amplitude images were analyzed toinvestigate the D-periodic axial gap/overlap spacing, chosen as our keymetric of fibril morphology (FIG. 12). Five to ten fibrils from eachaxial location were analyzed (SPIP v5.0.6, Image Metrology; Hørsholm,Denmark). Following image capture, a rectangular region of interest(ROI) was chosen along straight segments of individual fibrils (FIG.13). The ROI was drawn to ensure that it started and ended at the edgeof a gap zone, a method which minimizes edge effects that can degraderesolution. For each evaluated fibril, a two dimensional Fast FourierTransform (2D FFT) was performed and the primary peak from the 2D powerspectrum was analyzed to determine the value of the D-periodicgap/overlap spacing. This process decouples the measured D-Period repeatdistance from both pixel size and fibril orientation. A detailedanalysis of the uncertainty associated with this type of measurementsets the minimum bin size for population studies at 0.8 nm. For allhistograms, the bin size was therefore set at 1 nm.

All statistical analyses utilized SPSS (Version 16.0, SPSS Inc.). Forall investigations, a value of p<0.05 was considered significant. Toinvestigate differences in fibril morphology as a function of genotype,D-periodic axial spacing values measured from an individual sample werepooled, yielding an average value for that sample. The values from WT(n=4) and Brt1/+ (n=4) mice were then compared using One Way ANOVA.

To examine differences in the distribution of fibril morphology betweengenotypes, histograms and the Cumulative Distribution Function (CDF) ofeach group were computed. The CDF shows the fraction of a given samplewhich is contained up to a particular value, easily demonstratingdifferences between distributions in both mean and standard deviation.To test for statistically significant differences between distributions,Kolmogorov-Smirnov (K-S) tests were applied to the data. This test issensitive to changes in both the mean value and standard deviation of adistribution.

The D-periodic axial gap/overlap spacing was chosen as the key metric offibril morphology (FIG. 12). This measure captures aspects of fibrilstructure which may be related to the state of the individual moleculartriple helices, post-translational modifications and cross-linking. Toquantitatively assess the axial gap/overlap spacing in murine corticalbone as a function of OI genotype, femora from 2 month old male micewere used. In the intracortical bone along the anterior surface of thediaphysis, 9 locations were analyzed in each sample. Five to ten fibrilswere analyzed at each axial location. Within each genotype, measurementsat each axial location were pooled to assess the variability inmorphology along the length of the femur (FIG. 14). Qualitatively, thereis greater variability between axial locations in the Brt1/+ bones (B)in comparison to the WT (A) bones.

All measurements within each bone were pooled to yield the mean fibrilspacing for that bone (rightmost boxplot in each panel of FIG. 14, FIG.15). Within each genotype, the overall mean values were 67.6 nm and 67.4nm for WT and Brt1/+, respectively. There was no significant effect ofgenotype on mean fibril spacing (p=0.392) when the mean values from the4 bones in each genotype were compared by One Way ANOVA.

In addition to mean differences in morphology, important information canalso be obtained by viewing the population distribution of fibrilmorphologies within each genotype (FIG. 16). FIG. 16A shows that adistribution of spacings exists in each genotype and that thesedistributions differ between WT and dysplastic. The main population offibrils in each group was defined using the mean of the WT group ±1standard deviation, which is 66-70 nm (FIG. 16A). Within this range, 75%of WT, but only 55% of Brt1/+ fibrils, were found. As a second method tovisualize these distributions, the Cumulative Distribution Function(CDF) was computed from the measurements in each group (FIG. 16B). Boththe histogram and the CDF indicate that the Brt1/+ bones have apopulation of fibrils with closer spacing than does the WT group. Totest for statistically significant differences between distributions, afundamentally different comparison than the difference in means testedabove using One Way ANOVA, Kolmogorov-Smirnov (K-S) tests were appliedto the data. This test is sensitive to changes in both the mean valueand standard deviation of a distribution. The population distribution ofthe D-periodic spacings in Brt1/+ bones was significantly different thanthe WT bones (p=0.001).

In these experiments, surface characterization of Type I collagen at thenanoscale was linked with biology in an effort to understand theultrastructural mechanisms of an Osteogenesis Imperfecta (OI) phenotypein bone. Normal bone contains a distribution of Type I collagen fibrilmorphologies. As described herein, sheep subjected to two years ofovariectomy-induced estrogen depletion demonstrated a quantifiablechange in this distribution as a function of the disease state. However,the direct effects of estrogen-depletion on collagen are unknown and itis therefore difficult to understand why this change in the distributionof fibril morphologies exists. A major strength of the presentexperiments is that a disease system with a well-defined genetic changein the col1α1 allele and a well characterized alteration in collagenprotein structure was directly related to altered Type I collagennanoscale morphology. By characterizing the mean fibril spacing and thedistribution of fibril morphologies in the bone of normal mice and micethat were heterozygous for a specific col1α1 point mutation (G349C),phenotypic changes in the collagen fibril ultrastructure were detected.

Although the mean D-periodic spacing of Type I collagen fibrils inBrt1/+ bone was not significantly different than in WT, the distributionof spacing values was distinctive between the genotypes (FIG. 16).Further, there was more variation in fibril morphology along the axiallength of Brt1/+ bones (FIG. 14). In light of what is known about themutation in Brt1/+ mice and its detrimental effects on collagensynthesis, bone structure and overall bone mechanical integrity, thesefindings at the nanoscale are important. Bones from Brt1/+ mice havenormal levels of col1α1 and col1α2 transcripts, and produce Type Icollagen molecules with 3 different chain compositions molecules (2normal α1 chains, +/+; 1 normal and 1 mutant α1 chain, G349C/+; 2 mutantα1 chains, G349C/G349C) in an expected 1:2:1 ratio. Abnormal traffickingof collagen molecules with a single mutant chain leads to theaccumulation of collagen in the endoplasmic reticulum resulting indelayed secretion, over modification and selective degradation. As aresult, among the total collagen molecules secreted from Brt1/+ cells,the proportion with a single mutant chain ranges from 26-40%,considerably lower than the theoretical 50%. Once mutant molecules aresecreted from cells, they are efficiently incorporated into collagenfibrils and form crosslinks with the same efficiency as in WT mice.

Fibrils formed in Brt1/+ mice are thinner and exhibit a disruption ofthe normal quasi-crystalline lateral packing, but the methods used tomeasure these properties were indirect (N. V. Kuznetsova, A. Forlino, W.A. Cabral, J. C. Marini, S. Leikin, Structure, stability andinteractions of type I collagen with GLY349-CYS substitution in alpha1(I) chain in a murine Osteogenesis Imperfecta model, Matrix Biol. 23(2004) 101-112.). Data from the current study directly demonstrate thatthe incorporation of mutant molecules changes the intrafibrillarorganization of collagen fibrils. Changes in the D-period spacing arelikely driven by alterations in the end-to end spacing of collagenmolecules within the fibril due to changes in intracellulartropocollagen processing and assembly, or by a change in the tightnessof the twist of the fibril. Since collagen forms the template formineralization, altered collagen may also account for changes in mineralcomposition and density and may result in differences in therelationship between collagen and mineral. It is also possible thatchanges in the relationship between collagen, non-collagenous proteinsand mineral are partially responsible for the observed changes in fibrilmorphology.

Recent modeling studies have investigated the effects of point mutationson collagen structure and mechanical integrity (A. Gautieri, S.Vesentini, F. M. Montevecchi, A. Redaelli, Mechanical properties ofphysiological and pathological models of collagen peptides investigatedvia steered molecular dynamics simulations, J. Biomech. 41 (2008)3073-3077 and A. Gautieri, S. Uzel, S. Vesentini, A. Redaelli, M. J.Buehler, Molecular and mesoscale mechanisms of osteogenesis imperfectadisease in collagen fibrils, Biophys. J. 97 (2009) 857-865). In thefirst study, normal collagen helices and helices with single amino acidpoint mutations were modelled and the incorporation of these helicesinto triple helical collagen molecules was investigated. The majorconclusion from this study was that the alteration of the normal aminoacid sequence leads to packing differences in collagen molecules whichcould contribute to compromised mechanical integrity associated with OI.The current technology now directly observes that the alteration inamino acid sequence cause by a single point mutation in OI can lead tochanges in fibril spacing. Further investigation showed that OImutations can weaken intermolecular adhesions leading to decreasedstiffness and failure strength of affected collagen fibrils.Interestingly, those investigators also reported an increase inintermolecular spacing, which would be expected to lead to an increasein the D-periodic spacing of the resulting fibril. In the current directinvestigation of this phenomenon, mutant bone had a population offibrils with decreased spacing although no change in the average fibrilspacing was observed. An explanation for the disagreement between theseobservations is currently unknown. It is postulated that the decrease infibril spacing that was observed in the current study means that lessspace is available inside of the fibril for water and mineral to occupy.The decrease in water will have a direct negative impact on theviscoelastic and post-yield behaviour of the fibril. Further, as mineralis no longer formed within the template that the fibrils provide, anincreased proportion of extrafibrillar mineral will lead to the knownhypermineralization phenotype that is characteristic of OI.

In sum, the nano scale morphology of Type I collagen was probed to gaina mechanistic understanding of changes induced in the collagenultrastructure of bone in a murine model of Type IV OsteogenesisImperfecta. Although the average D-periodic gap/overlap spacing inBrt1/+ bone was the same as WT, Brt1/+ bone shows greater variability ofD-periodic spacing along the length of the bone and a significantdifference in the population distribution of collagen fibrils. Theseobservations at the nanoscale level provide insight into the structuralbasis for changes in bone composition, geometry and mechanical integrityin Brt1/+ bones. These changes in morphology may be directly related toalterations in nanoscale mechanical integrity.

The present technology further demonstrates that type I collagennanoscale morphology is similar in male and female mice. From studies ofinbred and congenic mouse strains and identification of quantitativetrait loci (QTLs) responsible for bone composition, geometric andmechanical properties, it is known that the genetic control of bone isgender specific. Gender-specific responses to genetic deficiency andmechanical loading have also been demonstrated. Differences exist inmusculoskeletal cells as a function of the gender of animal from whichthose cells were derived. The presence of these gender-specific skeletalphenotypes suggests that the efficacy of therapies designed to treatbone diseases may also be sex-specific.

Currently, little is known about gender-specific differences in boneextracellular matrix assembly and organization. Therefore, the presenttechnology and experiments were designed to investigate sex-specificdifferences in the nanoscale morphology of Type I collagen in bone. Itwas hypothesized that differences would exist in the mean D-periodicaxial gap/overlap spacing and population distributions of fibrilmorphologies as a function of gender in skeletally mature mice. However,the present study demonstrates that the average D-periodic gap/overlapspacing is not significantly different in male versus female boneswithin the ability of the present methods to detect a difference, andthere is no difference in the distributions of these values. Thesenanoscale observations suggest that although differences exist in thecells derived from male and female animals, gender-specific differencesin bone size, shape and mechanical integrity are likely derived athigher levels of the bone tissue hierarchy.

The following materials and methods were employed.

Mice from the C57BL/6J background strain were used with prior approval(University of Michigan, UCUCA protocol #09757). Four male mice and fourfemale mice were sacrificed at 16 weeks of age by CO₂ inhalation. Bothfemora from each mouse were harvested, and stripped of soft tissuebefore use.

Atomic Force Microscopy (AFM) imaging and analysis included thefollowing aspects. Before use, the proximal (above the third trochanter)and distal ends of each femur were removed using a low-speed sectioningsaw. The marrow cavity of each bone was cleaned using a small tubebrush. Bones were mounted, anterior-side up, to a steel disk using athin layer of cyanoacrylate glue. A flat surface of intracortical bonewas created using a 3 μm polycrystalline water-based diamond suspensionand a polished finish was created a 0.05 μm deagglomerated aluminasuspension (Buehler LTD; Lake Bluff, Ill.). Bones were sonicated for 15seconds to remove polishing residue and debris. To remove extrafibrillarsurface mineral and expose underlying collagen fibrils, each bone wasdemineralized using 0.5M EDTA at a pH of 8.0 for 15 minutes, thenvigorously rinsed with ultrapure water and soaked at 4° C. for at least16 hours. EDTA is often used in mineralized tissue research to removemineral while keeping collagen and cells intact and viable. Beforeimaging, each sample was briefly sonicated to remove any mineral thatwas still bound to the surface.

Samples were imaged in air using a PicoPlus 5500 AFM (Agilent). Imageswere acquired in tapping mode using silicon cantilevers (VistaProbesT300R, tip radius<10 nm, force constant 40 N/m, resonance frequency 300kHz; nanoScience Instruments; Phoenix, Ariz.) at line scan rates of 2 Hzor lower at 512 lines per frame. Making absolute x-y distancemeasurements with AFM has multiple limitations. Prior to sampling,calibration of the system was performed according manufacturerguidelines. This calibration led to a maximum tolerance of 1.1 nm.

Images were acquired from 9 axial locations in each bone sample(designated 1-9 beginning at the proximal end of the sample, FIG. 17).At each location, 3.5 μm×3.5 μm amplitude images were analyzed toinvestigate the D-periodic axial gap/overlap spacing, chosen as our keymetric of fibril morphology (FIG. 12). Five to ten fibrils from eachaxial location were analyzed as previously described. Briefly, arectangular region of interest (ROI) was chosen along straight segmentsof individual fibrils, starting and ending at the edge of a gap zone. Atwo dimensional Fast Fourier Transform (2D FFT) was performed and theprimary peak from the 2D power spectrum was analyzed to determine thevalue of the D-periodic gap/overlap spacing. A detailed analysis of theuncertainty associated with this type of measurement sets the minimumbin size for population studies at 0.8 nm. For all histograms, the binsize was therefore set at 1 nm.

All statistical analyses utilized SPSS (Version 16.0, SPSS Inc.). Forall investigations, a value of p<0.05 was considered significant. Toinvestigate differences in fibril morphology within each gender as afunction of axial location, the values at each location in each genderwere pooled and compared using One Way ANOVA with post hoc Bonferronitests. To investigate differences in fibril morphology as a function ofgender, D-periodic axial spacing values measured from an individualsample were pooled, yielding an average value for that sample. Thevalues from male (n=4) and female (n=4) mice were then compared usingOne Way ANOVA.

To examine differences in the distribution of fibril morphology betweengenotypes, histograms and the Cumulative Distribution Function (CDF) ofeach group were computed. The CDF shows the fraction of a given samplewhich is contained up to a particular value, easily demonstratingdifferences between distributions in both mean and standard deviation.To test for statistically significant differences between distributions,Kolmogorov-Smirnov (K-S) tests were applied to the data. This test issensitive to changes in both the mean value and standard deviation of adistribution.

To quantitatively assess the nanoscale morphology of Type I collagen inmurine cortical bone as a function of gender, femora from 16 week oldmale and female mice from the C57BL/6J background strain were used. Inthe intracortical bone along the anterior surface of the diaphysis, 9axial locations were analyzed in 4 femora from each gender. At eachaxial location, 70 μm×70 μm scans were performed to find suitable sitesfor closer inspection (FIG. 17). The scan size was decreased to a finalsize of 3.5 μm×3.5 μm. At each axial location, 5-10 fibrils wereanalyzed. In total, 259 fibrils from male samples and 263 fibrils fromfemale samples were analyzed.

By pooling data from the 4 bones in each gender at each of the 9locations and analyzing the D-periodic spacing, it was confirmed thatthere were no systemic changes in Type I collagen morphology as afunction of axial location in either gender (FIG. 18). The boxesrepresent the middle 50% of the data and the whiskers depict the dataextremes. The diamond is the mean and the line within the box is themedian of each group. The dashed horizontal line is for reference andcorresponds to the 67 nm axial spacing that is predicted by theHodge-Petruska model. Statistical comparisons were made between axiallocations within each gender using One Way ANOVA with post hocBonferroni tests. Although some statistical differences did existbetween locations in each gender (p<0.05; Male: 4 vs. 6; Female: 2 vs.9, 2 vs. 5, 8 vs. 9), there were no systematic differences in eithergender.

For each of the analyzed bones, the measurements within each bone werepooled to yield the mean fibril spacing for that bone. These mean valuesranged from 67.3 nm to 68.9 nm in males and 67.5 and 69.5 nm in females,with an overall mean value from the 4 samples measured in each gender of68.0 nm and 68.2 nm for males and females, respectively (FIG. 19). Whenthe mean values in each group were compared by One Way ANOVA, nostatistical differences were present between the genders.

FIG. 20 a demonstrates that a distribution of fibril morphologiesexisted in bones from each gender. Qualitatively, there were nodifferences present in the male and female population distributions(n=259 fibrils in males, n=263 fibrils in females). As a second methodto visualize the distributions, the Cumulative Distribution Function(CDF) was computed from the measurements in each group (FIG. 20 b). AKolmogorov-Smirnov (KS) test applied to the data indicates that thedistributions from the male and female bones were statisticallyindistinguishable from one another (p=0.276).

The present technology demonstrates that in cortical bone of skeletallymature mice, there are no differences in the D-periodic gap/overlapspacing of Type I collagen as a function of gender within the ability ofthis method to detect a difference. The D-periodic spacing of Type Icollagen was chosen as the metric to identify changes in nanoscalemorphology because it is readily quantified and is derived from thestructural arrangement of tropocollagen molecules within a collagenfibril (FIG. 12). The property is a fundamental characteristic of thisubiquitous structural protein and may be indicative of the state of theindividual molecular triple helices, post-translational modificationsand cross-linking. A previous study in male mice showed that adistribution of nanoscale morphologies (as measured by the D-periodicspacing) exists in Type I collagen-based tissues including bone, dentinand tendon. Further, collagen nanoscale morphology has been shown todiffer as a function of disease in bone. Findings in this study suggestthat although differences exist in the cells derived from male andfemale animals, gender is not a major factor that regulates thisfundamental characteristic of Type I collagen in bone.

Serum sex steroids (most notably estradiol, progesterone andtestosterone) are known to differ widely between male and female humansand mice, especially earlier in life. Notably, serum testosterone isroughly 20 times more abundant in males while basal estradiol andprogesterone are elevated by 2-4 times in females. The current studysuggests that these main hormonal differences between male and femalemice may not play a role in regulating Type I collagen nanoscalemorphology. However, as described herein, 2 years of estrogen depletionfollowing ovariectomy (OVX) resulted in dramatic changes in collagenmorphology in female sheep compared with sham operated control animals.In this case, the bones of these sheep developed under normal hormonallevels for 5 years. Following OVX in sheep, the production of female sexsteroids are dramatically reduced but not completely abolished. Othermetabolic changes may also be occurring following OVX. It seems probablethat a combination of factors that are not completely understood areresponsible for the changes noted in Type I collagen morphology.

At 16 weeks of age, mice are considered skeletally mature as mostmeasures of bone formation (both dynamic markers and size parameters)have reached a steady state value, peak bone mass has been attained andmechanical integrity has leveled off near a maximum level. At this age,a mouse is roughly equivalent to a human in their middle twenties. Miceof this age were used to avoid the possible effects of rapidlongitudinal and circumferential bone growth on collagen structure. At16 weeks of age, C57BL/6J mice have well known gender-specificdifferences in bone size, shape and mechanical integrity. Therefore, thecurrent study clearly indicates that in vivo, differences in Type Icollagen structure are not responsible for gender differences in tissuequality or mechanical integrity in these mice.

In sum, these experiments show that the D-periodic gap/overlap spacingof Type I collagen is not impacted by gender, within the ability of thepresent technology and methods to detect a difference. This observationat the nanoscale suggests that although differences exist in the cellsderived from male and female animals, gender-specific differences inbone size, shape and mechanical integrity are likely derived at higherorganizational levels of the bone tissue hierarchy.

The present technology can be used in several ways. For example, thepresent methods can be used to overcome the limited prognosticcapability of bone mineral density (BMD) analysis to diagnose subjectswith bone diseases (e.g., osteoporosis) or can be used to detect bonediseases at an earlier time or stage (e.g., prior to the onset ofclinical symptoms). The present methods can be used as screening anddiagnostic methods.

In some embodiments, the methods described herein can determine whethera subject has a bone disease, such as for example, osteoporosis andosteogenesis imperfecta. Such methods can include the steps of:providing a bone sample from the subject; determining a collagen fibrilmorphology value of the bone sample; providing a reference value from anon-affected control subject, wherein the reference value is a collagenfibril morphology value from the same type of bone sample obtained froma population of non-affected control subjects; and comparing thecollagen fibril morphology value of the bone sample to the referencevalue, wherein if the collagen fibril morphology value is altered versusthe reference value, the subject is said to have a bone disease.

In some embodiments, bone of a subject can be diagnosed or screened todetermine if the Type I collagen morphology is pathological andcorrelates to a collagen-related bone disease, for example, osteoporosisdisease and osteogenesis imperfecta among others. Other bone diseasesthat may be diagnosed or analyzed also include diseases of the bone andjoints or cartilage that involve defects in collagen formation orcollagen structure. In some embodiments, the subject's bone diseaseand/or other collagenous related diseases of tendons, skin, cartilage,or joints can be diagnosed and/or analyzed using cartilage samples fromtendons, skin, joints, and other collagen structures using biopsyprocedures.

In some embodiments, the present methods can be employed by firstproviding a bone sample from the subject. The bone sample can beobtained from the subject via biopsy or following a surgical procedure(e.g. joint replacement, dental extraction and the like). AFM can alsobe performed on live subjects by preparing a sample of tissue andexposing the subject's bone sample for further study. As used herein, abone sample can include any cortical or trabecular bone and partsthereof including the diaphysis, metaphysic, and epiphysis portions ofthe bone. In some embodiments, the bone sample can include part or thewhole tooth surface of one or more teeth, either extracted or in situ inthe mouth of the subject. For diagnosing and screening infants and youngchildren, deciduous teeth fall out during child hood can be used as abone sample in the methods described herein.

In some embodiments, an extracted portion of bone can be removed fromthe subject in order to determine a collagen fibril morphology valueusing AFM and 2-dimensional fast Fourier transformation (2D-FFT) toderive the collagen fibril spacing data obtained from the AFM images ofcollagen. In still other embodiments, a collagen fibril morphology valuecan be determined using AFM and 2D-FFT on the surface of one or moreteeth, in a non-invasive manner. Likewise, in some embodiments acollagen fibril morphology value can be determined using a subject'sskin. The skin may be from a biopsy or may be analyzed directly on thesubject.

In some embodiments, the collagen fibril morphology value is aphysiological feature of the subject's arrangement of type I, II, III orIV collagen in the subject's bone sample. It has been unexpectedly andsurprisingly found that the collagen fibrils contained on or within bonecan be used to determine whether the subject has a bone disease, asdescribed above, for example, osteoporosis and osteogenesis imperfecta,by determining the spacing and distribution of the fibrils imaged usingAFM and calculated using 2D-FFT. For each determination of the collagenfibril morphology value, for example, the fibril spacing and thedistribution of fibril spacing within a given bone sample, a 2D-FFT canbe performed on a flat plane surface of a bone sample. The 2D powerspectrum derived from the 2D-FFT can be analyzed to determine the valueof the D-periodic gap/overlap spacing, which is an example of a collagenfibril morphology value. The 2D-FFT analysis can yield data for allangular orientations and a sampling size of 9 D-period repeats, forexample, can be used to determine the fibril spacings. The method canalternatively include determining the variability in the fibril spacingsas a second illustrative example of a collagen fibril morphology value.Multiple analyzed sections of a given bone can provide a mean fibrilspacing for that bone; e.g., a tooth, tibia or femur.

In some embodiments, the method compares the subject's mean fibrilspacing for that tissue sample or type of bone and the variability ofthe fibril spacings determined by multiple measurements and compares toa population of mean fibril spacings for the same type of tissue or bonein subjects that are medically verified as not having the bone diseaseof interest. The reference value can be any quantitative collagen fibrilmorphology value, including as illustrative examples, mean fibrilspacing and fibril spacing variance for a given bone sample. Referencevalues can be determined in the exact same way as the subject's meanfibril spacing since AFM is a non-invasive technique and when combinedwith 2D-FFT for calculating the collagen fibril morphology value for thecontrol group not afflicted with the bone disease can be used todetermine the reference value.

In some embodiments of the present disclosure, the mean fibril spacingand/or variation on fibril spacing can be used as a collagen fibrilmorphology value to diagnose a subject with osteoporosis and other bonediseases or used as a basis for screening subjects that wish to knowwhether they are in fact suffering from disease. It has beenunexpectedly found that bones in two diseases, osteoporosis andosteogenesis imperfecta, have a significant impact on the distributionof fibril morphology that may indicate flaws in the collagen fibrilsfrom misalignment or over-modification, changes that can be compensatedfor over larger length scales. Other characteristics such as variabilityin the mean fibril spacing and fibril spacing themselves can be readyextrapolated from the AFM and 2D-FFT analysis of the subject's bonesample and correlate the experimentally determined collagen fibrilmorphology value with those of reference values for the same bone samplein healthy controls. If the subject's mean fibril spacing is alteredcompared with the reference value or if the distribution of fibrils isaltered in comparison to a reference distribution value, then thesubject can be presumptively diagnosed as having a bone disease and canbe confirmed with mineral density tests, for example.

In still other embodiments, diseases of the joint, tendons, skin, spineand other collagen related structures can also be diagnosed, analyzedand studied using the subject's mean fibril spacing and distributionpattern from collagenous tissue. If the subject's mean fibril spacingand distribution pattern is altered compared with the reference value orif the distribution of fibrils is altered in comparison to a referencedistribution value, the subject is presumptively diagnosed with adisease of the joint, cartilage or spine, for example. In someembodiments, the distribution in the mean fibril spacing when comparedto a reference value serves as a good diagnostic collagen fibrilmorphology value for the presence of a collagen related disease. Forexample, the large increase in the smaller D-spacing is a good indicatorof a bone disease. In other words, a large fraction of the populationshifting to lower values is correlated to the presence of a bonedisease, for example, osteoporosis and osteogenesis imperfecta.

In some embodiments, methods for identifying a subject as a diseasecandidate (the disease affecting tissue comprising collagen) includeusing scanning electron microscopy in place of atomic force microscopy,as described herein. For example, determining a collagen fibrilmorphology value of the tissue sample can include imaging the tissueusing scanning electron microscopy. D-periodic gap/overlap spacing of acollagen fibril can then be measured to provide the collagen fibrilmorphology value. The reference value can include a D-periodicgap/overlap spacing of a collagen fibril from the control subject. Insome cases, measuring D-periodic gap/overlap spacing of a collagenfibril to provide the collagen fibril morphology value further comprisesmeasuring D-periodic gap/overlap spacing of a plurality of collagenfibrils to provide the collagen fibril morphology value. Likewise, thereference value can further comprise a D-periodic gap/overlap spacing ofa plurality of collagen fibrils from the control subject. Comparing thecollagen fibril morphology value of the tissue sample to the referencevalue can then including comparing at least one of an overall mean and adistribution of the D-periodic gap/overlap spacing of the plurality ofcollagen fibrils.

Imaging using scanning electron microscopy can be performed as known inthe art. For example, a specimen typically needs to be dry as thespecimen chamber is at high vacuum. Hard and dry materials can beprepared relatively easily from collagenous tissues such as bone ordentin, which can be imaged with little further treatment. However,living cells and soft collagenous tissues, such as tendon and skin canrequire chemical fixation to preserve and stabilize their structure.Fixation, for example, can be performed by incubation in a solution of achemical fixative (e.g., glutaraldehyde, formaldehyde, among others) andcan include a postfixation treatment with osmium tetroxide. Fixed tissuecan then be dehydrated while trying to maintain structure. Becauseair-drying can result in collapse and shrinkage, it is commonly achievedby critical point drying, where water in the cells is replaced withorganic solvents such as ethanol or acetone, and replacement of thesesolvents in turn with a transitional fluid such as liquid carbon dioxideat high pressure. In the case of liquid carbon dioxide, the carbondioxide can be removed while in a supercritical state, so that nogas-liquid interface is present within the sample during drying. The dryspecimen can then be mounted using an adhesive, such as epoxy resin orelectrically-conductive double-sided adhesive tape, and sputter coatedwith a metal such as gold or gold/palladium alloy before imaging in themicroscope.

Sputter coating can be performed with a metal such as gold. Gold has ahigh atomic number and sputter coating with gold produces hightopographic contrast and resolution. However, the coating has athickness of a few nanometers, and can obscure underlying fine detail ofa specimen at very high magnification. Low-vacuum scanning electronmicroscopes with differential pumping apertures allow samples to beimaged without such coatings and without the loss of natural contrastcaused by the coating, but may not achieve the resolution attainable byconventional scanning electron microscopes with coated specimens.

When using a scanning electron microscope equipped with a cold stage forcryo-microscopy, cryofixation may be used and low-temperature scanningelectron microscopy performed on cryogenically-fixed tissue comprisingcollagen. Cryo-fixed tissue may be cryo-fractured under vacuum to revealinternal structure, sputter coated, and transferred onto the scanningelectron microscope cryo-stage while still frozen. For example,freeze-fracturing, freeze-etch, or freeze-and-break preparation methodsare available.

The following definitions and non-limiting guidelines should beconsidered in reviewing the description of the technology set forthherein. The headings (such as “Introduction” and “Summary”) andsub-headings used herein are intended only for general organization oftopics within the present disclosure, and are not intended to limit thedisclosure of the technology or any aspect thereof. In particular,subject matter disclosed in the “Introduction” may include noveltechnology and may not constitute a recitation of prior art. Subjectmatter disclosed in the “Summary” is not an exhaustive or completedisclosure of the entire scope of the technology or any embodimentsthereof. Classification or discussion of a material within a section ofthis specification as having a particular utility is made forconvenience, and no inference should be drawn that the material mustnecessarily or solely function in accordance with its classificationherein when it is used in any given composition.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

As used herein, the words “desire” or “desirable” refer to embodimentsof the technology that afford certain benefits, under certaincircumstances. However, other embodiments may also be desirable, underthe same or other circumstances. Furthermore, the recitation of one ormore desired embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the technology.

As used herein, the word “include,” and its variants, is intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices, and methods of this technology. Similarly, theterms “can” and “may” and their variants are intended to benon-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present technology that do not contain those elements orfeatures.

Although the open-ended term “comprising,” as a synonym ofnon-restrictive terms such as including, containing, or having, is usedherein to describe and claim embodiments of the present technology,embodiments may alternatively be described using more limiting termssuch as “consisting of” or “consisting essentially of.” Thus, for anygiven embodiment reciting materials, components or process steps, thepresent technology also specifically includes embodiments consisting of,or consisting essentially of, such materials, components or processesexcluding additional materials, components or processes (for consistingof) and excluding additional materials, components or processesaffecting the significant properties of the embodiment (for consistingessentially of), even though such additional materials, components orprocesses are not explicitly recited in this application. For example,recitation of a composition or process reciting elements A, B and Cspecifically envisions embodiments consisting of, and consistingessentially of, A, B and C, excluding an element D that may be recitedin the art, even though element D is not explicitly described as beingexcluded herein.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. Disclosures of rangesare, unless specified otherwise, inclusive of endpoints and include alldistinct values and further divided ranges within the entire range.Thus, for example, a range of “from A to B” or “from about A to about B”is inclusive of A and of B. Disclosure of values and ranges of valuesfor specific parameters (such as temperatures, molecular weights, weightpercentages, etc.) are not exclusive of other values and ranges ofvalues useful herein. It is envisioned that two or more specificexemplified values for a given parameter may define endpoints for arange of values that may be claimed for the parameter. For example, ifParameter X is exemplified herein to have value A and also exemplifiedto have value Z, it is envisioned that Parameter X may have a range ofvalues from about A to about Z. Similarly, it is envisioned thatdisclosure of two or more ranges of values for a parameter (whether suchranges are nested, overlapping or distinct) subsume all possiblecombination of ranges for the value that might be claimed usingendpoints of the disclosed ranges. For example, if Parameter X isexemplified herein to have values in the range of 1-10, or 2-9, or 3-8,it is also envisioned that Parameter X may have other ranges of valuesincluding 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9.

“A” and “an” as used herein indicate “at least one” of the item ispresent; a plurality of such items may be present, when possible.“About” when applied to values indicates that the calculation or themeasurement allows some slight imprecision in the value (with someapproach to exactness in the value; approximately or reasonably close tothe value; nearly). If, for some reason, the imprecision provided by“about” is not otherwise understood in the art with this ordinarymeaning, then “about” as used herein indicates at least variations thatmay arise from ordinary methods of measuring or using such parameters.

When an element or layer is referred to as being “on,” “engaged to,”“connected to” or “coupled to” another element or layer, it may bedirectly on, engaged, connected or coupled to the other element orlayer, or intervening elements or layers may be present. In contrast,when an element is referred to as being “directly on,” “directly engagedto,” “directly connected to” or “directly coupled to” another element orlayer, there may be no intervening elements or layers present. Otherwords used to describe the relationship between elements should beinterpreted in a like fashion (e.g., “between” versus “directlybetween,” “adjacent” versus “directly adjacent,” etc.). As used herein,the term “and/or” includes any and all combinations of one or more ofthe associated listed items.

What is claimed is:
 1. A method for identifying a subject as a diseasecandidate, the disease affecting tissue comprising collagen, the methodcomprising: providing a tissue sample from the subject, wherein thetissue comprises collagen; determining a collagen fibril morphologyvalue of the tissue sample; providing a reference value, wherein thereference value comprises a reference collagen fibril morphology valuedetermined using a tissue sample from a control subject, wherein thetissue sample from the control subject is the same type of tissue as thetissue sample from the subject; comparing the collagen fibril morphologyvalue of the tissue sample to the reference value; and identifying thesubject as a disease candidate when the collagen fibril morphology valueis different from the reference value, wherein determining the collagenfibril morphology value of the tissue sample comprises imaging thetissue; and measuring D-periodic gap/overlap spacing of a plurality ofcollagen fibrils to provide the collagen fibril morphology value;wherein measuring D-periodic gap/overlap spacing of a plurality ofcollagen fibrils to provide the collagen fibril morphology valuecomprises performing a two-dimensional Fast Fourier Transform for eachfibril and analyzing the primary peak from a resulting two-dimensionalpower spectrum to determine the D-periodic gap/overlap spacing value. 2.The method of claim 1, wherein the tissue sample comprises dentin, bone,tendon, or skin.
 3. The method of claim 1, wherein the tissue comprisestype I collagen.
 4. The method of claim 1, wherein providing a tissuesample from the subject comprises taking a biopsy of the tissue from thesubject.
 5. The method of claim 1, wherein the tissue sample isdemineralized using ethylenediaminetetraacetic acid.
 6. The method ofclaim 1, comprising imaging the tissue using atomic force microscopy. 7.The method of claim 1, wherein the reference value comprises aD-periodic gap/overlap spacing of a plurality of collagen fibrils fromthe control subject.
 8. The method of claim 1, wherein the collagenfibril morphology value is at least one of an overall mean and adistribution of the D-periodic gap/overlap spacing of the plurality ofcollagen fibrils.
 9. The method of claim 6, wherein imaging the tissueusing atomic force microscopy is performed in tapping mode.
 10. Themethod of claim 1, comprising: imaging the tissue using scanningelectron microscopy.
 11. The method of claim 1, wherein the subject andthe control subject are not the same sex.
 12. The method of claim 1,wherein the reference value comprises a reference collagen fibrilmorphology value determined using a plurality of tissue samples from aplurality of control subjects.
 13. The method of claim 12, wherein theplurality of control subjects includes a male control subject and afemale control subject.
 14. The method of claim 1, wherein the tissuesample comprises bone tissue and the disease comprises osteoporosis. 15.The method of claim 14, wherein: determining a collagen fibrilmorphology value of the tissue sample comprises: imaging the tissueusing atomic force microscopy; and measuring D-periodic gap/overlapspacing of a plurality of collagen fibrils to provide the collagenfibril morphology value, and the reference value comprises a D-periodicgap/overlap spacing of a plurality of collagen fibrils from the controlsubject; and identifying the subject as a disease candidate when thecollagen fibril morphology value is different from the reference valuecomprises: identifying the subject as an osteoporosis candidate when anoverall mean or a distribution of the D-periodic gap/overlap spacing ofthe plurality of collagen fibrils for the subject is different than thereference value from the control subject.
 16. The method of claim 1,wherein the tissue sample comprises bone tissue and the diseasecomprises osteogenesis imperfecta.
 17. The method of claim 16, wherein:determining a collagen fibril morphology value of the tissue samplecomprises: imaging the tissue using atomic force microscopy; andmeasuring D-periodic gap/overlap spacing of a plurality of collagenfibrils to provide the collagen fibril morphology value, and thereference value comprises a D-periodic gap/overlap spacing of aplurality of collagen fibrils from the control subject; and identifyingthe subject as a disease candidate when the collagen fibril morphologyvalue is different from the reference value comprises: identifying thesubject as an osteogenesis imperfecta candidate when the D-periodicgap/overlap spacing shows a greater variability along the length of thebone than the reference value from the control subject; or identifyingthe subject as an osteogenesis imperfecta candidate when the D-periodicgap/overlap spacing shows a difference in the population distribution ofcollagen fibrils compared to the reference value from control subject.18. A method of diagnosing an individual as having or not having adisease or condition affecting collagen by determining a morphologyvalue for collagen in the individual and comparing the morphology valueto that of a control subject, the method comprising determining themorphology value with a method comprising: providing a tissue samplefrom the subject, wherein the tissue comprises collagen; and determininga collagen fibril morphology value of the tissue sample; whereindetermining the collagen fibril morphology value of the tissue samplecomprises imaging the tissue; and measuring D-periodic gap/overlapspacing of a plurality of collagen fibrils to provide the collagenfibril morphology value; wherein measuring D-periodic gap/overlapspacing of a plurality of collagen fibrils to provide the collagenfibril morphology value comprises performing a two-dimensional FastFourier Transform for each fibril and analyzing the primary peak from aresulting two-dimensional power spectrum to determine the D-periodicgap/overlap spacing value.